database analytical methods rev 13

8/13/2019 Database Analytical Methods Rev 13

1/65

IIISSSP

PPF

FFO

OOR

RRE

EEN

NNS

SSI

IICCCB

BBI

IIOOOL

LLO

OOG

GGY

YY

DDDAAATTTAAABBBAAASSSEEEAAANNNAAALLLYYYTTTIIICCCAAALLLMMMEEETTTHHHOOODDDSSS

MMMAAANNNUUUAAALLL

Revision 13

5/23/12Issuing Authority: Quality Manager


2/65

Forensic Biology Database Analytical Methods Manual

Revision #13

APPROVED

May 23, 2012

Cynthia Cunnington

Forensic Scientist IVForensic Biology Supervisor/Technical Leader

I.

Revision 135/23/12

Issuing Authority: Quality Manager


3/65


REVISION RECORD

The following table must be filled out when revisions to the Biology Quality/Procedure Manual

are made.

Date: The effective date of the revision(s).

Revision #: The manual revision number.

Description: A brief description of the changes made to the manual.Addition: This column is checked if the revision reflects an addition (e.g. new SOP

or form) to the manual.

Deletion: This column is checked if the revision reflects a deletion (e.g. SOP orform no longer in use) from the manual.

Initials: Initials of the Technical Leader making the revisions.

Date Revision # Description Addition Deletion Initials

8/10/09 9(Rev. 1-8 part of

original singledocument. See

Quality Manual,

Appendix E)

Separated quality/casework methods/database

methods into three separate manuals, addedBSD/DNA IQ/Biomek 3000/PP16HS , and

Driftcon procedures and forms, fixed clerical

errors throughout

X CRH

11/29/10 10

Updated methods to reflect 3130 upgrade to

3130xl, clarified wording to agree with NDIS

procedures

CRC

8/29/11 11

Added offender thumbprint verification stamp

info and date in Tracker for criminal historyflag, updated offender sample storage location,

allowed for ILS at 50 RFU, removed print

instructions for Driftcon, fixed clerical errors

CRC

12/7/11 12

Separated out CODIS methods, removed

Quantifiler Human, added Plexor HY andquant/normalization/amp setup automation,

clarified contamination definition, clerical

X X CRC

5/23/12 13 Replaced 9947A with 2800M, clerical errors CRC

II. Revision 13

5/23/12Issuing Authority: Quality Manager


4/65


Table of Contents

i. Signature/Approval page

ii. Revision History

iii. Table of Contents

1-2 Individualization by STR Analysis

3-9 Offender Sample Receipt and Tracker Entry

10-17 Extraction Protocols

18-26 DNA Quantification: Real-Time PCR

27-34 STR Amplification: PP16 HotStart

35-57 STR Typing: Capillary Electrophoresis and Data Analysis

58-61 Driftcon FFC: Temperature Verification

III. Revision 135/23/12

Issuing Authority: Quality Manager


5/65

MBI-300

INDIVIDUALIZATION OF DNA SOURCES BY STR ANALYSIS

1.0 BACKGROUND:

Once a DNA source has been detected, and identified as to'source type' where applicable and feasible, it is oftenimportant to attribute the DNA sample to a particularindividual inasmuch as possible. Current DNA technology, inthe analysis of STR loci, offers individualization potential.However, the individualization of a particular sample occursthrough a comparative process. This process requires a DNAprofile from a known sample to which the evidence sampleprofile can be evaluated. Often times, this known sampleis identified through a search of the CODIS (either SDIS, orNDIS) database in cases where the perpetrator of theparticular crime is originally unknown. DNA samples arecollected from individuals convicted of qualifying crimesunder Idaho code and are then analyzed at STR loci for entryinto and subsequent searching against the DNA database.

2.0 SCOPE:

To provide uniform processing of DNA samples to achieve high

quality data and consistent interpretation.

3.0 EQUIPMENT/REAGENTS:

As listed in individual analytical procedures.

4.0 PROCEDURE:See Flow Chart on following page.

Database Analytical Methods: MBI-300Page 1 of 61

Revision 135/23/12

Issuing Authority: QualityManager


6/65

RECEIPT & DOCUMENTATION OFDATABASE SAMPLE

5.0 COMMENTS:

5.1 Careful scrutiny at each step will ensure insufficiencies

are identified, and compensated for where feasible, at theearliest possible point (see BI-318 for specifics).

ACCEPTABLE NOT ACCEPTABLE

ACCEPTABLE NOT ACCEPTABLE

EXTRACTION

BI-312

REVIEW & ENTRY INTOCODIS BI-302

AMPLIFICATIONBI-316

QUANTIFICATIONBI-314

CAPILLARY ELECTROPHORESISBI-318

GENEMAPPER ID ANALYSISBI-318

Database Analytical Methods: MBI-300Page 2 of 61

Revision 135/23/12



7/65

BI-301

OFFENDER SAMPLE RECEIPT AND DNA TRACKER ENTRY

1.0 BACKGROUND:

The implementation of the Combined DNA Index System (CODIS)in forensic DNA laboratories has provided an additional toolin assisting law enforcement agencies in solving or linkingcrimes that otherwise may not have resulted in theidentification of a suspect. It is important; however, thatsamples entered into the database be given a uniqueidentifier, which does not include any personal oridentifying information, in order to maintain the

confidentiality of the individual. Each laboratory mustdevelop a method of identifier assignment so that eachsample may be tracked and identified at a later time, if theneed arises.

Idaho Statutes: Title 19, Criminal Procedure, Chapter 55The Idaho DNA Database Act of 1996

ISP Forensic Biology Quality/Procedure Manual, Appendix C

2.0 SCOPE:

To provide a method for tracking offender database samplessubmitted for STR testing and CODIS entry, while ensuringindividual confidentiality.


Computer Workstation with ISP Intranet AccessBarcode EquipmentCourt Orders, Database Samples, and Report Forms

4.0 PROCEDURE:

4.1SAMPLE RECEIPT:

4.1.1Offender DNA samples and their corresponding DNACollection Report Forms received by the

Database Analytical Methods: BI-301Page 3 of 61

Revision 135/23/12



8/65

laboratory are to be marked with the date ofreceipt and the initials of the scientist whoreceived them. The sample and report form may bemailed to the laboratory separately; in the eventthat a sample has not been received, the

submitting agency should be notified.

Note: The Idaho Department of Corrections (IDOC)may submit an offender information printout inlieu of the collection report. The printout willbe treated the same as the report throughout theaccessioning process.

4.1.2Where possible, compare the DNA sample cardinformation to that of the Collection Report Formto ensure accuracy. Data for a sample may be

entered in absence of a DNA Collection Reportform. Additionally, an IDOC# is not necessaryfor data entry into DNA Tracker, but it ispreferred. Contact necessary IDOC personnel orsearch the corrections website(www.corrections.state.id.us) for a number if onehas not been recorded. The DNA Collection ReportForm will be retained with the sample after DNATracker data entry.

4.1.3The offense listed on the DNA sample card and/orCollection Report must be a qualifying offenseunder Idaho Code 19-5506 in order for the sampleto be entered into DNA Tracker and subsequentlyCODIS/NDIS. The corrections website listedabove, Idaho Court Repository, and/or ILETS willbe consulted for more information if the offenselisted is non-qualifying or unclear. Thescientist relies, in good faith, on theinformation provided by the submitting agency forentry into Tracker. It is not necessary, nor isit the scientists responsibility, to verify theoffense(s) of every Offender if the information

provided by the submitting agency qualifies aslisted. The ISP Attorney Generals Office willbe consulted on a case by case basis regardingthe release of information if a non-qualifyingoffender is involved in a candidate match.


Revision 135/23/12

http://www.corrections.state.id.us/http://www.corrections.state.id.us/


9/65

4.1.4Samples received in the laboratory that do nothave an associated qualifying offense will not beentered into DNA Tracker. The collection report(if present) will be marked to indicate that the

sample is a non-qualifying offense and returnedto the submitting agency. If there is nocollection report, a copy of the sample card willbe made, marked in the same manner, and returnedto the submitting agency. The sample(s) willthen be destroyed.

4.1.5 A thumbprint verification stamp will be placednear the thumbprint on the DNA sample card forall samples to be retained and entered into DNATracker (duplicate samples may be excluded). Thestamp will be used to record the thumbprintverification for hit confirmations and in orderto enter a DNA flag in the offenders criminalhistory. If no thumbprint is present, thesubmitting agency will be contacted and a newsample with print will be requested. It ispreferable to have a state identification number(SID) to facilitate the thumbprint verification.If no SID is listed on the card or report, thescientist should consult ILETS and record theSID, if found. The latent fingerprint sectionshould be consulted, as necessary, to determine

if a new sample and print needs to be requesteddue to insufficient print quality.

4.2COURT ORDER RECEIPT:

4.2.1Court order forms received by the laboratory areto be marked with the date of receipt and theinitials of the scientist who received them.

4.2.2The offense listed on the Court order must be a

qualifying offense under Idaho Code 19-5506 inorder for the court order information to beentered into DNA Tracker.


Revision 135/23/12



10/65

4.2.3If the offense on the court order is not aqualifying offense, no further action will betaken. The court order will be marked toindicate it is a non-qualifying offense andreturned to the submitting agency.

4.3 DNA TRACKER PRE-ENTRY SEARCH:

4.3.1Prior to data entry for any new sample or courtorder, a database search is performed toeliminate duplicate offender entry. Log on tothe DNA Tracker database program, located underForensics on the ISP Intranet.

4.3.2A duplicate offender search will be performedusing the Name field, followed by at least oneof the ID fields to maximize the potential forlocating an offender. Note: the DOB field mayonly be searched in combination with a name.

4.3.3If all of the searches return No matches found,the data for the new sample or court order may beentered as a New Offender (see 4.4).

4.3.4If a record(s) is returned that meets thecriteria, the data is examined and compared withthe new sample or court order received. If it is

determined that the Offender already exists inDNA Tracker, the new sample or court order andany additional Offender information will beentered under the appropriate tabs for thealready existing Offender. Each sample receivedfor an offender will be assigned a unique barcodenumber and will be retained in the laboratory(see 4.4).

4.4DNA TRACKER ENTRY:

4.4.1Enter basic Offender information from the courtorder, sample card, and/or DNA Collection Reportform as follows:


Revision 135/23/12



11/65

1)For Offenders not currently in Tracker, clickNew Offender at the top of the screen andfill in each of the appropriate fields with theOffenders primary information. Additionalinformation for Offenders already in Tracker

may be entered by clicking the Edit BasicDetails tab for that Offender.2)Verify all of the information is correct and

press the Save button in the top right cornerof the screen.

3)Add any additional alias names, DOBs, SSNs,and State Identification Numbers on theappropriate alias tab(s) that become availableafter saving the new offender. The SIDverification date will be filled in followingthumbprint/SID confirmation. Click the Savebutton after each entry.

4.4.2Enter each offense and its associated informationfrom the court order, sample card, and/or DNACollection Report form as follows:

1)Click the Offenses tab for the savedoffender.

2)Choose the appropriate offense from the pulldown menu and enter the correspondinginformation into the remaining fields.

3)Verify the information is correct and press theSave button in the top right corner of thewindow. Multiple offenses for the sameOffender must be entered and savedindividually.

4)Offense information may be updated/edited ifadditional data is received at a later time(e.g. when a sample arrives, fulfilling a courtorder or an additional sample for the sameoffense is received). Under the Offensestab, click on the appropriate offense codelisted in the table of offenses. Enter the

appropriate information and click the Savebutton.

4.4.3Enter Court Orders for an Offender as follows:


Revision 135/23/12



12/65

1) Click the Court Orders tab for the savedoffender.

2)Enter the court order issue and received dates.Note: the order received date willautomatically populate in the sample history,

once a sample has been received fulfilling thatcourt order.3)Click the Add Offense link and choose the

appropriate offense/court case from the pulldown menu. Note: the offenses in the pull downlist are those previously entered in theOffenses tab. A court order must have anassociated offense in order to be entered;therefore, the offense information must beentered prior to the court order.

4)Verify the information is correct and click theSave button.

5)Once the court order has been entered it isretained in the laboratory.

4.4.4Enter samples for an Offender and print samplebarcodes as follows:

1)Click on the Samples tab for the savedOffender.

2)Enter the sample information into theappropriate fields, leaving the barcode fieldblank (Tracker will automatically generate a

unique barcode number if the field is notfilled in).

3)If the submitting agency is not listed in theagency pull down menu, it may be added byclicking on the edit agencies button to theright of the agency field.

4)If the sample received is pursuant to apreviously entered court order, click on theShow Unfulfilled Court Orders link. Clickthe box next to the appropriate court order tomark it as fulfilled by the sample being

entered.5)Enter the number of barcode labels to be

printed. One label is to be placed on the DNAsample card/folder, one on the collectionreport, and one inserted behind the FTA card


Revision 135/23/12



13/65


Revision 135/23/12


envelope for placement on the FTA card at thetime of analysis.

6)Verify the information is correct and click theSave button. Barcode labels willautomatically print upon saving the sample

information.7)Staple the DNA Collection Report (if present)to the inside of the DNA sample folder andattach the appropriate barcode labels. Thelabeled DNA sample card, with collectionreport, will be stored in the DNA DatabaseLaboratory.

8)Barcode labels may be printed at a later timefor individual samples, by clicking on theappropriate sample from the Samples tab orfor multiple samples, by using the BatchSamples function at the top of the screen.


14/65

BI-312

EXTRACTION PROTOCOLS FOR PCR DNA TYPING TESTS

1.0 BACKGROUND:

Many methods exist to obtain DNA, suitable for amplification,from a variety of sources. It has become increasingly importantto streamline the extraction process through automation. Avariety of extraction methods and robotics systems have becomeavailable for the successful extraction and downstream processingof known database DNA samples, using both blood and buccalcollection methods. The Promega DNA IQ Extraction System and theBeckman Coulter Biomek 3000 robotic workstation are both examplesof systems that have proven successful in the forensic community.

Comey, CT et al. DNA Extraction Strategies for AmplifiedFragment Length Polymorphism Analysis. J For Sci, Vol. 39,1994, pp. 1254-1269.

McLaren, B et al. Automating the DNA IQ System on the Biomek3000 Laboratory Automation Workstation. Profiles in DNA, 2006Vol. 9(1): 11-13

Tereba, A et al. High-Throughput Processing of Samples on SolidSupports Using the SlicPrep 96 Device. Profiles in DNA, 2005Vol. 8(2): 3-5.

DNA IQ System-Database Protocol, Technical Bulletin (revisiondate 4/06), Promega.

Automated DNA IQ System Protocol for the Beckman Coulter Biomek3000, Technical Bulletin (revision 1/11), Promega.

2.0 SCOPE:

To provide appropriate protocols for the extraction of databasesample DNA, both manual and automated, suitable for PCR

amplification and subsequent analyses.

3.0 EQUIPMENT:

Pipettes and tipsHarris PunchBSD600-Duet Semi-Automated Sample Punch Instrument w/ Scanner

Database Analytical Methods : BI-312Page 10 of 61

Revision 135/23/12



15/65

96-well Reaction Plate and BaseMicroAmp Tubes1.5mmicrocentrifuge Tubes (1.5mtubes)65C Oven

SlicPrep DeviceAdhesive Foil Seal70C Water Bath or Heat Block with SlicPrep AdaptorCentrifuge with Swinging Plate RotorBiomek 3000 Robotics System with MagnaBot 96 Magnetic Separation

Device, 1/4 Inch Foam Spacer, Variomag Teleshake, HeatingBlock, and Tip Disposal Container

Reagent Reservoirs and Frame1.2 mRound-Well, Deep Well Plate

4.0 REAGENTS:

FTA Purification ReagentPCR TE (TE, 10mM Tris-HCl; 0.1mM EDTA, pH 8.0)DNA IQ System Reagents99% Isopropyl Alcohol95-100% Ethanol1M Dithiothreitol (DTT)

5.0 DNA EXTRACTION PROCEDURES:

5.1 FTA EXTRACTION FROM OFFENDER DATABASE SAMPLES:

Note: Since the DNA remains bound to the FTA card, regular,non-filter pipette tips may be used throughout and asingle tip may be used for each reagent. A multi-channel pipettor and 96-well reaction plate may be usedfor larger sample batches.

5.1.1 Remove 1-3 punches" from the FTA card using a 1.2mmHarris punch (this is accomplished by placing punchfirmly on card and twisting 1/2 turn clockwise and 1/2turn counterclockwise). Eject/punch sample(s) intomicroAmp tube(s)/plate. Alternatively, the BSD puncher

may be used to punch 1 2mm punch directly into theplate (see 5.2.1).

5.1.2Add 150FTA reagent to microAmp tube(s)/plate, gentlymix and incubate at RT for ~5 minutes.

5.1.3Remove and discard FTA reagent from sample(s).


Revision 135/23/12



16/65

5.1.4Repeat 5.1.2-5.1.3twice.

5.1.5 Add 150TE to microamp tube(s)/plate, gently mix andincubate at RT for ~5 minutes.

5.1.6Remove and discard TE from sample(s).

5.1.7Repeat 5.1.5-5.1.6twice.

5.1.8 Make sure the punch is at the bottom of the microAmptube(s)/plate, using a sterile pipette tip ifnecessary. Place tubes/plate, uncovered in 65C ovenfor 2 hours.

5.1.9Proceed to PCR Amplification (see BI-316).

5.2 AUTOMATED DNA IQ EXTRACTION FROM OFFENDER DATABASE SAMPLES

5.2.1 SAMPLE PUNCHING USING THE BSD600-DUET

5.2.1.1 Fill out the BSD/Biomek Load Sheet on theBSD Load Sheet tab of the Databaseworksheets template(Form 312-BI). Print acopy for the batch record/file. Choose theBSD Input File tab and ensure theinformation is correct and corresponds to theLoad Sheet information entered. Delete anyunused wells, making sure to keep End File

as the last cell. Perform a Save As of theworksheet to disc (e.g. USB drive) forsubsequent transfer to the BSD600 puncher.The document must be saved as a .txt file.Save the template as samples areautomatically populated on the other tabs forsubsequent processing steps.

Note: The last two columns of the plateshould be left empty for control placement insubsequent steps. This allows for 78

samples, an extraction control, and a reagentblank to be tested per plate.

5.2.1.2 Ensure the BSD tray table is adjustedproperly by placing the appropriate plates inboth positions and adjusting the height, asnecessary. The bottom of the chute should be


Revision 135/23/12



17/65

~2mm from the top of the plates, measured ateach corner.

5.2.1.3 Turn on the BSD puncher and computer and plugin the air/pressure pump. Make sure water

is present in both filter bottles (~50-100ml). Double click the BSD Duet MainMenu icon and login using the appropriateuser name and password.

5.2.1.4 Choose Configure Systemand open the Filestab. Ensure that File Inputis selected fromthe Sample Number Modedropdown list. Clickthe Browsebutton and locate your savedinput file to fill in the Input File Namebox. Click Save and Exit.

Note:Using the input file option onlyallows for a single plate to be punched at atime. To punch multiple plates, the processmust be repeated for each new set ofsamples.

5.2.1.5 Choose Distribute Spotsand click Continue.When prompted to select test group to punch,highlightAll Available Testsand clickContinue. Check the box next to InputFile.tstand check the samples box on the

right. Press Continue. This test file willpunch 3 of the 3.2mm spots (verify beforebeginning). If punching for FTA extraction,the number and size of punches will need tobe changed under the Edit Test Sequencesoption prior to beginning.

5.2.1.6 Place the appropriate plate into position 1of the BSD tray table with well A1 locatedin the upper left corner. Click Continuetobegin punching. When using the SlicPrep 96

Device, remove the white collar, push thebasket down into the plate, and set thecollar aside before processing.

5.2.1.7 Scan the sample barcode using the scannerattached to the BSD and place the card underthe card clamps on top of the instrument.Use the light guide dots to properly position


Revision 135/23/12



18/65

the card for punching. Press the foot switchwhen ready to punch or use the delay switchfor automatic punching. Repeat until allsamples have been punched.

Note:The scanned barcode must match that onthe input file or the instrument will not

punch the card.

5.2.1.8 Remove the plate and proceed to SlicPrep pre-processing (see 5.2.2) or FTA extraction (see5.1).

5.2.2 SLICPREP PRE-PROCESSING

5.2.2.1 Add 400ul DNA IQ Lysis Buffer with DTT (see5.2.3.1) to each well of the device plate.Seal the top of the plate with a foil sealand place into a 70C water bath or heatblock with SlicPrep adaptor for 15-60 min.

5.2.2.2 Without removing the foil, raise the filterbasket and place the white collar back ontothe device by sliding it under the basket andaligning the detents on the device.

5.2.2.3 Centrifuge the device at 1500 x g (~3000RPM)for 5 min. in a swinging plate rotor.

Remove and discard the collar and filterbasket upon completion of centrifugation.

5.2.2.4 Proceed to DNA IQ Extraction on the Biomek3000 (see 5.2.4).

5.2.3 DNA IQ REAGENT PREPARATION

5.2.3.1 When opening a new DNA IQ kit, add 1.5ml 1MDTT to the bottle of Lysis Buffer (1ulDTT/100ul lysis buffer) and mix. Mark the

bottle with initials, the date the DTT wasadded, and the DTT lot number. Theactivated Lysis buffer may be used for upto 2 months following DTT addition.Alternatively, the lysis buffer may beprepared as needed based on the number ofsamples to be processed.


Revision 135/23/12



19/65

5.2.3.2 Prepare the new wash buffer (70ml 2X bottle)by diluting with 35ml Isopropyl Alcohol and35ml Ethanol for a final 1X concentration.Mark the bottle with initials, the date ofalcohol addition, and the alcohol lot

numbers. The 1X wash buffer may be used fora period of 1 month after activation.Alternatively, the wash buffer may beprepared as needed based on the number ofsamples to be processed.

5.2.3.3 Prepare/aliquot each of the DNA IQ reagentsat the time of use, based on the followingcalculations and reservoir positions(instructions listed in the ReagentPreparation comments window):

1: Elution Buffer = 2.5ml + (#samples x100ul) 80 samples = 10.5mlNote: be careful not to contaminate the

elution buffer with lysis buffer or other

reagents

2: Lysis Buffer w/ DTT + Resin = [860ul +(#sample columns x 344ul)lysis] + [140ul +(#sample columns x 56ul)resin]80 samples = 4.3ml lysis + 0.7ml resin

3: 1X Wash Buffer = 1.5ml + (#sample columnsx 2.4ml) 80 samples = 25.5ml

4: Lysis Buffer w/ DTT = 2.0ml + (#samplecolumns x 0.8ml) 80 samples = 10.0ml

5.2.4 DNA IQ EXTRACTION USING THE BIOMEK 3000 ROBOTICS

SYSTEM

5.2.4.1 Turn on the Biomek 3000, computer, and Watlowheater. Make sure that the heater is set to85oC and that the shaker electrical box dialis turned to the red at the far left stop.

5.2.4.2 Open the Biomek Software and choose theB3K_DNAIQV2.0.1.ISP method.

5.2.4.3 Set up the Biomek 3000 deck as follows(outlined in the Beckman Hardware and DeckSetup comments window):


Revision 135/23/12



20/65

Rack 1 (A1): Tool rack with MP200 (1)andgripper (3)

ML1 (A2): P250 barrier pipette tipsML2 (A3): P250 barrier pipette tips

(dependent on sample number)

ML3 (A4): P250 barrier pipette tips(dependent on sample number)ML4 (A5): emptyP6 (A6): Heating systemP1 (B1): PCR plate and baseP2 (B2): Deep round-well purification plateP3 (B3): Reservoir frame and reservoirs with

reagentsP4 (B4): Deep round-well purification plateP5 (B5): MagnabotP7 (B6): Pre-processed deep square-well plate

stacked on the Variomag Teleshaker

Note: The number of tip boxes will depend onthe number of samples to be extracted. Theinstrument will only extract full columns onthe plate.

5.2.4.4 Click the green run instrument arrow buttonto begin the run. When prompted, enter thenumber of columns to be extracted (endcolumn), the first tip column, and either50ul, or 100ul for elution volume. The 50ul

volume may be preferable for lowerconcentration samples. Verify the reagentand deck layout and click ok.

5.2.4.5 When the run has finished remove thePCR/elution plate. A brief spin may benecessary to remove any bubbles present inthe wells.

5.2.4.6 Proceed to realtime PCR (BI-314) forquantification of the purified DNA obtained

in 5.2.4.5.

5.2.4.7 Clean the reservoirs between runs. At thecompletion of all runs for the day, wipe downthe deck with 10% bleach or Dispatchsolution. Tools may also be cleaned with 70%Ethanol if necessary.


Revision 135/23/12



21/65


Revision 135/23/12


6.0 DNA EXTRACTS:

6.1After a plate of samples has been extracted and during

subsequent analyses (e.g. quantification and amplification),the DNA extracts may be stored sealed with foil atapproximately 4oC. For longer storage periods, the extractsshould be frozen at approximately 20oC. These extracts arein-progress work product during this stage(s).

6.2 Any extract remaining, following the successful completion ofanalysis and data review, will be discarded.

7.0 Comments:

7.1 A reagent blank shall be carried through all extraction stepsto check the purity of the reagents being used. There needonly be one reagent blank per extraction plate, but theanalyst may choose to run additional blanks on a given plate.

7.2The analyst may optionally incorporate cleaning punches duringBSD sample punching. At a minimum, the puncher is cleanedmonthly but may be cleaned more often at the analystsdiscretion.

7.3 These procedures may be used for blood or buccal samples onFTA paper.

7.4The DNA IQ resin must be thoroughly mixed/resuspended prior toaliquoting.

7.5The final elution volume attained on the Biomek 3000 will be~8-10ul less than the volume chosen, due to evaporation duringthe processing.

7.6The DNA IQ extraction procedure may be performed manually, ifnecessary, following the procedure outlined in the DNA IQSystem-Database Protocol, Technical Bulletin (revision date

4/06). This procedure follows the same steps as performed onthe Biomek 3000.


22/65

BI-314

DNA QUANTIFICATION: REAL-TIME PCR

1.0 BACKGROUND:DNA methodologies that employ the PCR, such as STR analysis,necessitate consistent quantification of human DNA to obtainoptimum data.

"Developmental Validation of a Real-Time PCR Assay for thesimultaneous Quantification of Total Human and Male DNA," Krenke,B.E., et al, Forensic Science International: Genetics 3 (2008), pp14-21 Vol. 3.

Improving Efficiency of a Small Forensic DNA Laboratory:Validation of Robotic Assays and Evaluation of Microcapillary ArrayDevice, Crouse, C., et al, Croat Med J 2005, Vol. 46, No. 4, pp.563-577.

Plexor HY System for the Applied Biosystems 7500 and 7500 FASTReal-Time PCR Systems, Technical Manual (revision 11/07), PromegaCorporation.

Automated Plexor HY System Setup for the Biomek 3000, TechnicalBulletin (revision 1/10), Promega Corporation.

7500/7500 Fast Real-Time PCR Systems Maintenance Guide, AppliedBiosystems.

2.0 SCOPE:To provide a reliable method for the consistent quantification ofvariable amounts of human DNA isolated from database samples.


ABI 7500/Computer 96-well Reaction Plate/BaseABI 7500 SDS Software Optical Adhesive CoverPlexor Analysis Software Microcentrifuge TubesPipettes and Tips Strip Tubes/CapsPlate Clamp Reservoir frame


Automated Tube Holders Centrifuge

Revision 135/23/12



23/65

Plexor HY Kit PCR-TEBiomek 3000 Robotics System with MagnaBot 96 Magnetic Separation

Device, 1/4 Inch Foam Spacer, Variomag Teleshake, HeatingBlock, and Tip Disposal Container

4.0 PROCEDURE:

4.1 MANUAL PREPARATION OF DNA STANDARDS:

4.1.1Label 8 sterile microcentrifuge tubes, or strip tubesA through H or 1-8.

4.1.2Dispense 40of PCR-TE into tubes B-H (Std. 2-8).

4.1.3Mix the Plexor HY Male Genomic DNA Standard thoroughlyby vortexing approximately 5 seconds. Transfer 10to tube A (Std. 1/undiluted) and to tube B (Std. 2).Mix the dilution thoroughly.

4.1.4Prepare Std. 3-7 via a serial dilution by mixing andsubsequent 10transfers from tubes B through G. Thedilution series consists of 50, 10, 2, 0.4, 0.08,0.16, 0.0032ng/(or adjusted per kit QC), and a no-template control, respectively.

4.2MANUALREACTION PREPARATION:

4.2.1Determine the number of samples to be quantified

(including, at minimum, 2 sets of DNA standards).

4.2.2Finish filling out the 7500 Load Sheet on the Databaseworksheets template (Form 312-BI). Print a copy for thebatch record/file.

4.2.3 Calculate the volume of reaction components needed,based upon the number of samples to be quantified andadding 2 or 3 reactions to compensate for loss andvariability due to pipetting. The following are thevolumes needed per reaction.

Plexor HY 2X Master Mix 10Plexor HY 20X Primer/IPC Mix 1Water 7

Note: The volume of reaction components necessary toprepare the Master Mix will be automatically


Revision 135/23/12



24/65

calculated upon Load Sheet data entry and choosingthe manual option.

4.2.4 Thaw the master mix and primer mix and vortex 3-5seconds. Pulse-spin prior to opening the tube (do not

overspin).

4.2.5 Place a 96-well reaction plate into a base, being carefulnot to touch the top or individual wells. Do not placethe plate directly onto the counter or any surface otherthan its base or the ABI 7500 thermal block.

4.2.6 Prepare the Master Mix by pipetting the required volumesof water, primer and master mixes into an appropriatelysized microcentrifuge tube or reservoir. Mix thoroughlyand pulse spin if mixing in a tube.

4.2.7 Carefully pipette 18of the PCR Reaction Mix into thebottom of each reaction well to be used. Blowing-outthe pipette is not recommended to avoid splashing and/orbubbles in the well.

4.2.8 Add 2of sample or standard to the appropriatereaction well, being careful to avoid bubbles as much aspossible.

4.2.9 Seal the reaction plate with an Optical Adhesive Cover.Proceed to 4.4.

4.3 AUTOMATED REAL-TIME PCR SETUP USING THE BIOMEK 3000 ROBOTICS

SYSTEM:

4.3.1 Finish filling out the 7500 Load Sheet on the Databaseworksheets template (Form 312-BI). Choose the automatedoption and print a copy for the batch record/file.

4.3.2 Thaw the master mix and primer mix and vortex 3-5seconds. Pulse-spin prior to opening the tube (do not

overspin).

4.3.3 Turn on the Biomek 3000 and computer. Open the BiomekSoftware and choose the B3KPlexorHY_V1.0.1.ISPmethod.

4.3.4 Set up the Biomek 3000 deck as follows (outlined in theLabware Requirements comments window):


Revision 135/23/12



25/65

Rack 1 (A1): Tool rack with MP200 (1), P200L (2), andgripper (3)

ML1 (A2): P250 barrier pipette tipsML2 (A3): P50 barrier pipette tips

ML3 (A4): P50 barrier pipette tips (dependent on tipquantity)ML4 (A5): emptyP6 (A6): Heating system (not used)P1 (B1): PCR plate and base with extracted DNAP2 (B2): Plate clamp and base with strip tubes in

positions 1 and 12P3 (B3): Reservoir frame with automated tube holders at

left-most and right-most positionsP4 (B4): PCR plate and baseP5 (B5): Magnabot (not used)P7 (B6): Variomag Teleshaker (not used)

Note: The position 1 strip tubes will be used forpreparation of the standard dilution series. Ifpreparing standards manually or using previouslyprepared standards, these will be placed in position 1.

4.3.5 Place reagent tubes in the position P4 tube holders asfollows (outlined in the Tube Holder Positions andReagents comments window):

Left 1: Plexor HY 2X Master Mix

Left 2: Plexor HY 20X Primer/IPC MixLeft 3: EmptyLeft 4: Empty 1.5ml microcentrifuge tubeRight 1: Amplification Grade WaterRight 2: PCR-TE Buffer in 1.5ml tubeRight 3: Plexor HY Male Genomic DNA StandardRight 4: Empty

Note: The empty microcentrifuge tube will be used forthe master/reaction mix preparation. If preparing thereaction mix manually, it will be placed in this

position. The left positions 1 and 2 will be empty ifusing manually prepared reaction mix. Right positions2 and 3 will be empty if using manually or alreadyprepared standards.

4.3.6Click the green run instrument arrow button to beginthe run. When prompted, enter Y or N for standards andmaster mix preparation, the number of samples to be


Revision 135/23/12



26/65

quantified, the first available P250 tip, and the firstavailable p50 tip. Verify the reagent and deck layoutand click ok.

4.3.7When the run has finished remove the PCR plate and seal

with an Optical Adhesive Cover. A brief spin may benecessary to remove any bubbles present in the wells.Proceed to 4.4.

4.3.8At the completion of all runs for the day, wipe downthe deck with 10% bleach or Dispatch solution. Toolsmay also be cleaned with 70% Ethanol if necessary.

4.4RUNNING THE REACTION:

4.4.1Turn on the 7500 computer and login with the appropriateuser name and password. After the computer hascompletely started up, power on the 7500 instrument,allowing it to warm up at least ~30 seconds. Launch theABI 7500 SDS Software.

4.4.2Open the instrument tray by pushing on the tray door.Place the plate into the tray holder so that well A1 isin the upper-left corner and the notched corner of theplate is in the upper-right corner.

4.4.3 Close the instrument tray by gently pushing the rightside of the tray door.

4.4.4 In the SDS software, select File>Newand chooseAbsoluteQuantitationfor Assay, 96-Well Clearfor Container, andPlexor HYfor Template.

4.4.5 Highlight the wells that contain samples or standardsand apply the autosomal, Y, and IPC detectors. Do notinclude any unused wells. It is not necessary to namethe samples.

Note: Detectors are created during the initial

instrument set-up and/or kit usage. Refer to theQuantifiler Kits Users Manual (page 2-11) forinstructions on creating detectors if needed.

4.4.6 Select the Instrumenttab and review the thermal cyclerconditions [Stage 1: 1 cycle, 95oC, 2:00 min.; Stage 2:38 cycles, 95oC, 00:05 min, 60oC, 0:35 min.; Stage 3: 1cycle, 95oC, 00:15 min, 60oC, 1:00 min, 95oC, 00:15 min;


Revision 135/23/12



27/65

20sample volume; 9600 emulation unchecked; Datacollection: Stage 2, Step 2 (60.0@0:35)]

4.4.7 Save the plate document as a .sds file with theappropriate plate name.

4.4.8 Under the Instrumenttab, click Startto begin the run.When the run has completed, proceed to 4.5.

Note: Choose yes at the SYBR Green message prompts.

4.5 ANALYSIS AND RESULTS:

4.5.1Open the plate document to be analyzed.

4.5.2SelectAnalysis>Analyze.

4.5.3Export the amplification data as a .csv file (e.g. toUSB drive) by selecting File>Export>Delta RN.

4.5.4 Export the melt/dissociation data as a .csv file (e.g.to a USB drive) by selecting File >Export >Dissociation>Raw and Derivative Data. Select Yes at the SYBRGreen message prompt.

4.5.5 Launch the Plexor Analysis Software and deselect SetPassive Reference On Import in the file menu.

4.5.6Import the .csv files by selecting File>Import New Run.Enter the plate (assay) name, choose the AppliedBiosystems 7500 instrument and assign dyes to theautosomal (FLR), Y (CO560), and IPC (CR610) targets.Check the amplification and melt boxes for each andclick next.

4.5.7Enter the analysts initials under Operator Name andselect next. Select the appropriate amplification andmelt files using the browse buttons and clickfinish.

4.5.8Select the Sample IDs tab and enter the names for eachwell. Alternatively, the 7500 Load Sheet may be copiedand pasted into the Sample IDs window using the ctrl-Tfunction.

Note: sample names (including standard and no-templatecontrols) must be unique or the software will report


Revision 135/23/12



28/65

concentration averages and an error will occur whenimporting data into the worksheets.

4.5.9Define the unknown samples, no-template controls, andstandards by highlighting the appropriate wells in the

well selector pane of the PCR Curves tab and clickingthe corresponding icon in the toolbar.

4.5.10Highlight the standard wells and click the CreateDilution Series icon in the tool bar. Select verticalseries and decreasing. Enter 50 (or adjusted valuefrom the kit QC) as the starting concentration and 5 asthe dilution factor. Click apply.

4.5.11Leave the standard wells highlighted and in the meltcurves window, drag the target melt temperature line tothe midpoint of the melt curves. Do this for theautosomal, Y, and IPC curves. The melt temperature (Tm)for each sample will be displayed in the table on theright.

A yes or no in the Tmcolumn indicates whether asample has a melt temperature within the target range.A no call indicates that the sample has a melt curvewithin the range but there is insufficient sample tocross the threshold. An increase in the Tm for unknownsmay indicate the presence of impurities.

4.5.12With the standard wells still highlighted, select theautosomal channel and click the Add Standard Curveicon in the tool bar. Repeat for the Y channel. Selectthe Standard Curves tab to view the standard curvesand print the autosomal screen for the batch record.Review the autosomal data for inconsistencies from thefollowing:

An R2value of >0.990 indicates a close fit between thestandard curve regression line and the individual CTdata points of quantification standard reactions.

An R2value of


29/65

The slope should fall within the typical slope range of3.2 to 4.0. A slope of 3.3 indicates 100%amplification efficiency.

4.5.13Under the PCR Curves tab, highlight all of the

samples, standards, and no-template controls (if anystandards were omitted in 4.5.12, do not include themhere). Select the Add Standard Curve icon for theautosomal and Y channels. Click ok to replace theexisting standard curves.

4.5.14Open the forensics report by selecting Forensics >SetNormalization and IPC Parameters >OK. Check the IPC andCurves Status columns for potential problems andtroubleshoot as necessary (viewing other report tabs ifdesired). Unknown samples with IPC Ctvalues severalcycles higher than those of DNA standards with similarconcentrations, is an indication that inhibition mayhave occurred.

4.5.15Highlight and copy the forensics report. Paste the datainto the PHY Raw Data tab of the database worksheets.Open the PHY 7500 Results tab and review the importeddata. Delete any unused wells from the sheet.

If performing automated normalization and amplificationset-up follow the instruction on the sheet, delete thestandards and no-template controls and print the results

sheet for the batch record. A .tab file willautomatically be saved for Biomek 3000 import.

If performing manual normalization and amplificationset-up choose the check mark from the manual dilutionscheme drop down menu and un-gray the associatedcolumns. Delete the standards and no-template controlsand adjust values in the dilution scheme columns asneeded. Print a copy of the results sheet for the batchrecord.

4.5.16Proceed to STR amplification (BI-316).

5.0COMMENTS:

5.1After initial thawing, the Plexor HY Male Genomic DNA Standardwill be stored at approximately 4oC.


Revision 135/23/12



30/65


Revision 135/23/12


5.2 Refer to the Plexor HY System Technical Manual for additionaluser information and troubleshooting guidelines.

5.3 A template may also be created in the Plexor AnalysisSoftware when running multiple plates with the same layout.

5.4 The Halogen Lamp may be checked manually to determine ifreplacement is needed. Place the Green Calibration Tray inthe block. Select Instrument > Calibrateand set theexposure time to 4096ms, lamp control toMax, and selectFilter A. Click Snapshot and observe results. Expectedresults should consist of red fluorescence displayed in allwells. Lack of fluorescence indicates the need for lampreplacement. The lamp status should be checked as well byselecting Instrument > Lamp Status/Replacementand viewingthe condition.

5.5 In order to extend the life of the Halogen Lamp, theinstrument should be turned off anytime it is not in use.Lamp life is approximately 2,000 hours.


31/65

BI-316

STR AMPLIFICATION: PP16 HS

1.0 BACKGROUND:

DNA analyses have revolutionized forensic biology. Theadvent of PCR allowed scientists to analyze evidentiarymaterial present in minute quantities and degraded states.The identification of forensically significant STR loci hasallowed scientists to combine the discrimination attainablewith the older RFLP technology with the speed and samplingcapabilities of other PCR-based methodologies. ThePowerPlex 16 HS allows the co-amplification of the coreCODIS 13 loci, as well as, Amelogenin, and twopentanucleotide-repeat loci, Penta D and Penta E.

Butler, J. Forensic DNA Typing: Biology and TechnologyBehind STR Markers.(2001) Academic Press.

PowerPlex 16 HS System Technical Manual, PromegaCorporation

2.0 SCOPE:

To provide a reliable method for consistent, high qualityamplification of DNA from offender database samples ensuringthe generation of suitable PCR product for capillaryelectrophoresis and analyses of these STR loci.


Thermalcycler Half reservoir and baseCentrifuge Automated tube holdersMicrocentrifuge tubes PCR plate and base1.1ml square-well, v-bottom plates Pipettes and tips

Foil Seal/Optical Adhesive Cover Compression padPowerPlex 16 HS KitBiomek 3000 Robotics System with MagnaBot 96 Magnetic

Separation Device, 1/4 Inch Foam Spacer, VariomagTeleshake, Heating Block, and Tip Disposal Container


Revision 15/23/1

Issuing Authority: QualityManage


32/65

4.0 PROCEDURE:

4.1DNA TEMPLATE:

4.1.1Based upon the quantity of DNA isolated and its

initial concentration, samples should be normalized toan optimal concentration for amplification (e.g.,0.25ng/for manual normalization). It is alsoconvenient to have all samples in a batch at the sameconcentration, if possible, for ease in thepreparation of PCR Master Mix and reaction additions.The maximum amplification volume for low level orundetected samples is 4.4for manually preparedPowerPlex 16 HS, quarter volume reactions and 4.0for automated reactions.

4.1.2The amount of DNA template and 2800M added to aquarter volume amplification reaction should betargeted at ~0.50ng, but may be adjusted as necessary.Additionally, a higher amount of the positive controlis necessary for FTA samples due to the loweramplification cycle number.

4.2MANUAL AMPLIFICATION SET-UP:

4.2.1Thaw the PowerPlex 16 HS 5X Master Mix, PowerPlex 16HS 10X Primer Pair Mix, and Amplification Grade Water(optional) contained in the HotStart kit.

4.2.2Calculate the volume of reaction components neededbased upon the number of samples (including extractionand amplification controls) to be amplified and adding1 or 2 reactions to compensate for loss andvariability due to pipetting. Use the Amp.Worksheet tab of the Database Worksheets Template(Form 312-BI) and the manual setup option toautomatically calculate necessary volumes. Print acopy of the completed worksheet for the batchrecord/file. The following is a list of the 'fixed'

amounts to be added for a 6.25reaction.

PowerPlex 16 HS 5X Master Mix 1.25PowerPlex 16 HS 10X Primer Pair Mix 0.61DNA Template + dH2O 4.4


Revision 135/23/12



33/65

1For FTA extracted samples there is no volume for theDNA template so 4.4of dH2O will be added to thesetubes/wells.

Note: The amount of Reaction Mix added to each sample

is dependent on the volume needed to add the DNAtemplate.

4.2.3Prepare the reaction mix by pipetting the requiredvolumes of 5X master mix, 10X primer mix, and water(if needed) into an appropriately sizedmicrocentrifuge tube or reservoir. Mix thoroughly andpulse-spin if using a tube.

4.2.4Pipette the appropriate amount of reaction mix intoeach well of a labeled 96-well reaction plate. Thenegative amplification control should be the lastsample processed.

4.2.5If DNA concentrations were not the same, addappropriate volume of dH2O as necessary.

4.2.6Pipette each DNA sample into the appropriate well.Use 2800M control DNA for the positive amplificationcontrol and dH2O for the negative amplificationcontrol.

4.2.7Thoroughly seal the plate with an adhesive foil seal.

Use a seal applicator to tightly seal between and overeach well.

4.2.8Remove lab coat and, touching only the plate,transport the samples to the thermal cycler in theAmp/PostAmp room, using the other hand on the doorknob.

4.2.9Place a compression pad over the plate and put it intothe thermal cycler. Remove/discard gloves beforeexiting the Amp/PostAmp room. Proceed to 4.4.

4.3AUTOMATED AMPLIFICATION SET-UP USING THE BIOMEK 3000 ROBOTICSSYSTEM:

4.3.1 Thaw the PowerPlex 16 HS 5X Master Mix and PowerPlex 16HS 10X Primer Pair Mix contained in the HotStart kit.


Revision 135/23/12



34/65

4.3.2 Finish filling out the Amp Worksheet on the databaseworksheets template (Form 312-BI). Choose the automatedoption and print a copy for the batch record/file.

4.3.3Turn on the Biomek 3000 and computer. Open the BiomekSoftware and choose the B3KNormSTR_V1.0.0.ISP method.

4.3.4 Set up the Biomek 3000 deck as follows:

Rack 1 (A1): Tool rack with P20 (1), P200L (2), andgripper (3)

ML1 (A2): P250 barrier pipette tipsML2 (A3): P50 barrier pipette tipsML3 (A4): P50 barrier pipette tipsML4 (A5): P50 barrier pipette tips (dependant on tip

count)P6 (A6): Heating system (not used)P1 (B1): PCR plate and base with extracted DNAP2 (B2): Dilution plate (square-well, v-bottom)P3 (B3): Reservoir frame with automated tube holders at

left-most and right-most positions and halfreservoir in the center

P4 (B4): Dilution plate (square-well, v-bottom)P5 (B5): PCR plate and base (magnabot removed)P7 (B6): Variomag Teleshaker (not used)

4.3.5 Place reagents in the position P3 tube holders and

reservoir as follows:

Left 1: PP16HS 5X Master MixLeft 2: PP16HS 10X Primer Pair MixLeft 3: EmptyLeft 4: Empty 1.5ml microcentrifuge tubeHalf Reservoir: autoclaved nanopure waterRight 1: 1ng/ul 2800M DNA StandardRight 2: EmptyRight 3: EmptyRight 4: Empty

Note: The empty microcentrifuge tube will be used forthe master/reaction mix preparation. If preparing thereaction mix manually, it will be placed in thisposition. The left positions 1 and 2 will be empty ifusing manually prepared reaction mix.


Revision 135/23/12



35/65

4.3.6Click the green run instrument arrow button to beginthe run. When prompted, enter the first available P250tip, the first available p50 tip, and true/false formanual master mix preparation.

4.3.7The STR Normalization Software will open automatically.Login with the appropriate user name and password.

4.3.8Enter the plate name and select Idaho State Police QtrRxn 16HS 0.5ng Plexor as the template and clicknext.

4.3.9Select use an existing extraction control template(Idaho State Police Database Ext Control Template) andchoose next.

4.3.10Choose use existing injection control template andselect Idaho State Police Inj Control Template. Clicknext.

4.3.11Click browse to locate the .tab input data filecreated in step 4.5.15 of BI-314. Select next andreview the analysis breakdown table.

4.3.12Click review extraction plate and make any necessarychanges (e.g. manually add or remove samples foramplification). Select done and next.

4.3.13The amplification plate may be reviewed by selectingreview amplification plate. Samples will be re-ordered on the plate to fill any blank spaces createdby removing samples. Select done and next.

4.3.14Select the appropriate CE results group and instrumentprotocol, ensure open report is checked, enter theappropriate user name and password, and click finish.Select ok for the P20 tool message.

4.3.15Verify the deck layout and reagent setup and click

ok.

4.3.16When the run has finished remove the PCR plate andthoroughly seal the plate with an adhesive foil seal.Use a seal applicator to tightly seal between and overeach well.


Revision 135/23/12



36/65

4.3.17Remove lab coat and, touching only the plate,transport the samples to the thermal cycler in theAmp/PostAmp room, using the other hand on the doorknob.

4.3.18Place a compression pad over the plate and put it intothe thermal cycler. Remove/discard gloves beforeexiting the Amp/PostAmp room. Proceed to 4.4.

4.3.19At the completion of all runs for the day, wipe downthe deck with 10% bleach or Dispatch solution. Toolsmay also be cleaned with 70% Ethanol if necessary.

4.4THERMAL CYCLING PARAMETERS:

4.4.1After the samples have been placed in the thermalcycler, turn on the power and select the appropriatepre-programmed cycling profile.

4.4.1.1For DNA IQ-extracted and low level FTA-extracted samples, use 'pp16HS' with thefollowing conditions:

96C for 2 minutes, then:

ramp 100% to:94C for 30 seconds,

ramp 29% to:60C for 30 secondsramp 23% to:70C for 45 secondsfor 10 cycles, then:

ramp 100% to:90C for 30 secondsramp 29% to:60C for 30 secondsramp 23% to:

70C for 45 secondsfor 20 cycles, then


4C soak


Revision 135/23/12



37/65

4.4.1.2For FTA-extracted samples (other than lowtemplate), PP16HS17 with the followingconditions may be used:


ramp 100% to:94C for 30 seconds,ramp 29% to:60C for 30 secondsramp 23% to:70C for 45 secondsfor 10 cycles, then:

ramp 100% to:90C for 30 secondsramp 29% to:60C for 30 secondsramp 23% to:70C for 45 secondsfor 17 cycles, then


4C soak

5.0AMPLIFIED DNA PRODUCT:

5.1 After cycling has concluded remove samples from thermalcycler. Samples should be run on the Genetic Analyzer assoon as possible after amplification. Prior to capillaryelectrophoresis and/or before analysis is completed thesamples may be stored at approximately 4C. For longerstorage periods, samples should be frozen at approximately -20C. Amplified product is ONLYstored in the Amp/PostAmproom.

5.2 At a point in time after STR analysis is completed (i.e.,

data has been reviewed and approved for upload), theamplified product will be disposed of in a biohazardcontainer in the amp/post-amp room. As needed, thiscontainer will be sealed and transported directly to thedishwashing room. The container will be placed into a secondbiohazard bag, sealed and disposed of with other biohazardousmaterial.


Revision 135/23/12



38/65


Revision 135/23/12


6.0 COMMENTS:

6.1Clean surfaces with freshly made 10% bleach solution orDispatch prior to set-up.

6.2Wear gloves at all times during amplification set-up.

6.3 Mix all reagents thoroughly (e.g., vortex) and pulse-spinthem in microfuge prior to dispensing.

6.4If excessive evaporation is observed followingamplification, an optical adhesive cover may be used to sealthe plate rather than the foil seal.

6.5When using a volume reaction, the amplification reagentsmay be completely absorbed by the 2mm BSD punch, if used forthe FTA extraction method. If this happens, it isrecommended that 5ul sterile water be added post-amplification, and thoroughly mixed prior to running on thegenetic analyzer.

6.6After initial thawing the 2800M DNA standard and anydilutions will be stored at approximately 4oC.

6.7When amplified product is stored in the refrigerator, an

increased amount of condensation may form on the foil seal.Punching through the foil, following a brief spin, may bepreferable to removing the seal in order to reduce thepotential for contamination.


39/65

BI-318

STR TYPING: CAPILLARY ELECTROPHORESIS AND DATA ANALYSIS

1.0 BACKGROUND:

Any eukaryotic genome is interspersed with repeated DNA sequencesthat are typically classified by the length of the core repeatsequence, and the range of contiguous repeats typically seen orthe overall length of the repeat region. STR (Short TandemRepeat) loci are scattered throughout the genome occurring every10,000 nucleotides or so, and have core repeat units of 2-6bp inlength with overall lengths of less than 400 bp.

STR loci examined for human identification purposes were selectedfor the following characteristics: 1) high discriminating power(generally >0.9) with observed heterozygosity of >70%, 2) loci onseparate chromosomes to avoid linkage, 3) ability to obtainrobust, quality, reproducible data when multiplex amplification isperformed, 4) low stutter, 5) low mutation rate and 6) smallallele sizes (


40/65

Butler, J. Forensic DNA Typing: Biology and Technology BehindSTR Markers.(2001) Academic Press.

PowerPlex 16 HS System Technical Manual

PowerPlex Matrix Standards, 3100/3130, Technical Bulletin

ABI 3130/3130xl Genetic Analyzer Getting Started Guide

ABI 3130/3130xl Genetic Analyzer Mainenance Troubleshootingand Reference Guide

GeneMapper ID Software User Guide

2.0 SCOPE:

To provide a reliable method for generating STR geneticprofiles from offender DNA database samples.


3130XL Genetic Analyzer with Data Collection SoftwareGeneMapper ID SoftwareComputers9700 Thermal CyclerPipettes and TipsBenchtop CoolerCapillary Arrays

96 Well Reaction Plates and SeptaBuffer Reservoirs and SeptaPOP4 PolymerGenetic Analyzer BufferPowerPlex 16 HS Kit ContentsPowerPlex 16 Matrix StandardsDeionized FormamideNanopure Water

4.0 PROCEDURE:

4.1AMPLIFIED FRAGMENT DETECTION USING THE 3130xl

Note:Prior to using the ABI 3130xl Genetic Analyzer forsamples, a spectral calibration (matrix standards) must berun to achieve proper color separation of the dyes used forthe amplification primers, allelic ladders and sizestandard. To prepare a matrix, four standards are rununder the same capillary electrophoresis conditions that


Revision 15/23/1



41/65

will be used for samples and allelic ladders. Use the 3130Matrix Standard set, which includes the Fluorescein Matrix,JOE Matrix, TMR Matrix and CXR Matrix for the blue, green,yellow and red matrix standards, respectively. This isperformed when necessary due to performance, or after any

instrument maintenance/repair that involvesadjustment/replacement of the CCD camera or laser.

Additionally, a Spatial Calibration must be performed priorto running any samples. The instrument uses imagescollected during the spatial calibration to establish arelationship between the signal emitted for each capillary,as well as the position where that signal falls and isdetected by the CCD camera. This is performed any time acapillary is installed or replaced (including temporaryremoval of a capillary) or if the instrument is moved.

4.1.1Turn on the computer, turn on the instrument,start Data Collection Software and wait for greensquares to appear for all applications on theservice console. Expand the necessary subfolderson the left tree pane of Data Collection. Referto the ABI 3130/3130xl Genetic Analyzers GettingStarted Guide for detailed instructions oninstrument set-up (including creation ofinstrument protocols, results groups, and spatialcalibration). Fill-in appropriate information inthe 3130/3130xl Injection Log (Form 422B-QC).

Shut down is performed in the opposite order(Data Collection software, 3130xl, thencomputer). The Data Collection Software must beclosed by choosing Stop All and waiting for allred symbols to appear before closing. Never usethe X to close while green or yellow symbolsare displayed.

4.1.2 Create a new plate record:

4.1.2.1 For a spectral calibration plate expandthe tree pane of the Data CollectionSoftware and click Plate Manager, underga3130xl. Choose New, and fill inthe dialog boxes, with SpectralCalibration as the application. Fill inthe applicable dialog boxes on the


Revision 15/23/1



42/65

Spectral Calibration Editor as follows(clicking OK when complete to save):

Sample Name:date_Spectral

Priority: May optionally be changed to anumber


43/65

GeneMapper: GeneMapper_Generic_Instance

Sample Name:

Allelic Ladder: LADDER (or PP16_LADDER)

Controls: POS [or (+), etc.], NEG [or (-), etc.], RB (reagent blank)

Database samples: ID##########(e.g., ID2001001412).

Priority: May optionally be changed to anumber


44/65

samples .50ng (injection timesmay be adjusted [3-10 secondsper analyst's discretion).

5 secs.- Samples


45/65

Value of On, and click Send Command. Next,in the Command Name box, choose Set oventemperature, with a Value of 60.0 and clickSend command. Note:once the oven has beenturned on and the temperature set, the oven will

only preheat for 45 minutes before shuttingitself off.

4.1.4 Prepare samples for capillary electrophoresis:

4.1.4.1 For amplified products (includingcontrols), typically 1-1.5rxn isadded to 10of ILS Master Mix (made byadding 0.5ILS600 size standard/sample;9.5deionized formamide/sample andadding quantities for N+2 in Master) thathas been dispensed into the wells of apre-labeled plate. For Allelic Laddersadd 1Ladder to 10Master Mix. Note:The master mix may be altered by adding0.25, 0.5, or 0.75ILS600 sizestandard to 9.759.5, or 9.25deionized formamide respectively toaccount for ILS peak height variability.

4.1.4.2 Matrix samples are diluted 1:10 inNanopure H2O (a 1:5 dilution may benecessary). 5of each matrix dye

fragment is then added to 480ofdeionized formamide (without sizestandard). Load 25 of the fragmentmix into each of 16 wells on the pre-labeled plate, which will include each ofthe 16 capillaries (e.g. wells A1 throughH1 and A2 through H2).

4.1.5Following sample addition, place a plate septa onthe plate and heat denature for ~3 minutes at95C. Immediately chill in benchtop cooler (or

on ice) for 3 minutes (perform on all sampletypes - ladders, matrix, controls and samples).Note:the plate septa may be cut to cover onlythose well columns being used on smaller plateruns.


Revision 15/23/1



46/65

4.1.6 Place the sample plate into the plate base andsecure the plate retainer clip on top, makingsure that no gray is visible through the holes.

4.1.7Place the plate assembly in the instrument in

either position A, or position B and close thedoors. The plate map on the Plate View window,under Run Scheduler will turn yellow when theplate is in place and has been detected by theinstrument. Note:the 3130xl will accommodatetwo plates per run.

4.1.8Prior to running the plate, confirm that dye setF is selected and the correct active calibrationfor dye set F is set in spectral viewer.

4.1.9 Locate the plate record in the Plate Viewwindow and highlight it by clicking on it once.With the plate record highlighted, click theappropriate plate map (position A or position B)to link the plate to that specific record. Theplate map will turn from yellow to green when itis successfully linked. Verify the correctscheduling of the run in the Run View window.Select a run and confirm that the correspondingwells highlighted in the plate diagram arecorrect for that run. Make adjustments to theplate record if necessary.

4.1.10Click the green Run Instrument arrow button inthe toolbar to start the run. Monitorelectrophoresis by observing the run, view,array, or capillaries viewer window. Eachinjection (set of sixteen samples) will take ~45minutes. Note:to run a duplicate plate record,the plate may need to be unlinked prior tolinking the duplicated record. This is done byhighlighting the currently linked plate recordand clicking unlink.

4.1.11After completion of the spectral calibration run,open the Spectral Viewer window to evaluate thespectral and set the active calibration. Confirmthat Dye Set F is selected. Click on individualwells in the plate diagram to see results foreach of the sixteen capillaries. For eachcapillary, verify that four peaks are present in


Revision 15/23/1



47/65

the spectral profile (upper pane), that the orderof the peaks are, from left to right, blue-green-yellow-red, and that the peaks are regular inappearance. Next verify that four peaks arepresent in the raw data profile (lower pane),

that the order of the peaks are, from left toright, red-yellow-green-blue, and that the peakheights are above 750RFU (1,000-4,000 RFU isideal). If at least 12 capillaries pass, thenthe calibration should be saved and set as theactive calibration.

Note: A minimum of 12 of the 16 capillaries mustpass in order to accept a spectral calibration.A passing capillary will be colored green in theplate diagram. Additionally, capillary statusmay be viewed in the Event Log underInstrument Status. Rerun the spectralcalibration as necessary until at least 12capillaries pass.

4.1.12 After completion of the run finish filling outthe 3130xl Injection Log (Form 422B-QC).

4.2DATA ANALYSIS: GENEMAPPERID (GMID)

4.2.1 Data analysis is NOT performed on the instrument

computers. Transfer the run folder (includingthe 3130xl plate record) to an analysis computerusing a portable USB drive. After analysis andreview are complete, a copy of the run folder andGMID project(s) will be stored on the ISPFSnetwork drive. The Run Folder on the instrumentcomputer may be deleted at this point.

Note:prior to data analysis, the appropriatepanels and bins must be imported into GeneMapper

ID. Additionally, previously run Macintosh data

must first be converted to PC files using theMac to Win conversion program.

4.2.2 Set up the analysis methods for GMID analysis asfollows (analysis methods are created and storedin the Analysis Methods tab in GeneMapperManager):


Revision 15/23/1



48/65

GeneralTab:Name the analysis method so that itreflects what the method is (e.g. 3130PP16-150RFU).

Allele Tab:Choose the appropriate bin set.

Choose Use marker-specific stutter ratio ifavailable, and ensure minus stutter distancesare from 3.25 to 4.75 for tetra and from 4.25 to5.75 for penta. All others should be 0.

Peak Detector Tab:Advanced Peak DetectionAlgorithm, partial sizing (80-550 or 600), lightsmoothing, Local Southern size calling methodwith baseline window of 51 pts, min. peak halfwidth = 2, polynomial degree = 3, peak windowsize = 15, and slope thresholds = 0.

Analysis range may be set to either full orpartial and is empirically determined for eachrun and/or instrument. When using partial range,the start and stop points are determined by areview of the raw data and choosing points thatwill not include the primer peaks but will coverthe size range of 80 to 500 bases.

Peak Amplitude Thresholds will depend on samplequality. Generally 150 rfu threshold in allcolors. Rfu threshold may be raised in Blue,

Green and Yellow for Allelic Ladders ifnecessary. Rfu threshold may be lowered to 100at the analyst's discretion. With the exceptionof Red, peaks below 100 rfu are deemedinconclusive (see 4.3.2RFU Threshold).

Peak Quality Tab:The minimum peak height ratiofor Heterozygote Balance should be set at 0.5 fordatabase (CODIS) samples. Set the max peak widthto 1.5 bp and pull-up ratio to 0.05. The signallevel and allele number may be set according to

analyst preference and sample type.

Quality Flags Tab:The quality flags are onlyused as a tool to aid in data analysis and review(i.e. to assist in calling attention to potentialartifacts or data quality concerns). These flagsettings may be adjusted according to analystpreference and sample quality.


Revision 15/23/1



49/65

4.2.3 Create and store a size standard for GMIDanalysis, under the Size Standards tab inGeneMapper Manager. Name the size standard sothat it reflects what the standard is (e.g.

ILS600 80-600).

Data analysis will be performed using the Basicor Advanced size standard. The size standardconsists of the following peaks: 60, 80, 100,120, 140, 160, 180, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 550, and 600(the 60 and 600 peaks may be optionally definedby the analyst).

4.2.4 Create a GeneMapperID Project:

4.2.4.1 From the GMID main menu, select File/AddSamples to project. Highlight theappropriate run folder in the pop-upwindow and click Add to List. Once therun folder has been copied to the columnon the right, click Add to populate theproject with the samples in the runfolder.

4.2.4.2In the Samples table, for each sample,select the sample type, analysis method,

panel, and size standard from the pull-down lists. Ladders must be assigned thesample type of Allelic Ladder for theanalysis to occur. In order to use thecontrol concordance quality flag, allcontrols must be marked appropriately aseither Positive Control, or NegativeControl. All others may be marked asSample.

4.2.4.3Save the project as the date MMDDYY,

followed by the analyst' initials (andany other descriptors that may benecessary). Note:the analyzed projectwill be exported to the run folder at thecompletion of analysis/review.

4.2.4.4Analyze the samples by clicking the greenAnalyze button. If the project has not


Revision 15/23/1



50/65

already been saved, a prompt will appearto enter a project name before analysiswill commence.

4.2.5Evaluate GeneMapperID Data:

4.2.5.1The Raw Data may be reviewed to determineanalysis start/stop points, or toidentify baseline problems, off-scaledata, excessive 'spikes' or otheranomalies that may interfere with dataanalysis and require re-injection orother corrective measures. Expand therun folder located in the navigation paneon the left. Highlight the sample(s) ofinterest to view the associated sampleinformation, raw data and EPT data.Minimize or highlight the run folder toreturn to the main project window.

4.2.5.2Check the SQ (sizing quality) for allsamples. A green square indicates thatthe sample has passed the sizing criteriaand need not be manually examined.Examine the size standard of each samplewith yellow and/or red SQ to confirmcorrect assignment of fragment sizes.Highlight the sample(s) of interest and

click the Size Match Editor button. Ifnecessary, adjust the peak assignments byright clicking on a peak and deleting,adding, and/or changing values. If allpeaks are correctly labeled but thequality score is below 1.0 (may bechecked by choosing Tools/Check SizingQuality), click the Override SQ buttonto set the SQ to 1.0. Once all editshave been made, click OK to save thechanges and close the Size Match Editor

(clicking Apply saves the changes butleaves the Size Match Editor open).These samples are ready for reanalysis inthe project window.

Note:Data may still be deemed acceptablewithout the ILS 60 and/or 600 bp peakspresent. If additional peaks are


Revision 15/23/1



51/65

assigned because of bleed-through of TMRpeaks (typically Amelogenin peaks), thescientist may choose an Analysis Method,with an increased rfu threshold for thered channel to prevent these peaks from

being detected, if desired.

4.2.5.3Examine the blue, green, and yellowallelic ladders. Check that correctallelic assignments were made.

Note:GMID automatically averages allvalid ladders in a run for genotyping.Genotypes are assigned by comparing thesizings of unknown alleles from sampleswith the sizings of known allelescontained within the averaged allelicladders of each locus. A ladder(s) maybe omitted from analysis by deleting itfrom the main project window prior toanalysis.

4.2.5.4 Data may be examined in variouscombinations of colors and/or tables toidentify bleed-through, spikes, stutter,-A, off-ladder variants, etc. SamplePlots viewed from the Samplestab/window, allows all loci in a given

color(s) to be viewed simultaneously.The Sample Plots view from theGenotypes tab/window; however, allowsloci to be viewed individually (more thanone locus can be viewed at the same timeby adjusting the number of panesdisplayed).

4.2.5.5GeneMapperID includes a series ofquality flags (PQVs) to alert the analystof potential sample quality concerns. A

green square indicates that sample datahas passed all of the quality checks, butyellow or red indicate that the data hasa problem with one or more of the qualitychecks. A yellow or red flag does notnecessarily mean that the data is bad orunusable and the flags are not to berelied on solely. The analyst may choose


Revision 15/23/1



52/65


53/65

4.2.5.10 Samples demonstrating an off-ladder ( smallest or largest ladder allele,respectively), tri-allele, ormicrovariant (alleles with incomplete

repeats) allele(s) shall be re-analyzedfor verification (reinjection may besufficient for off-ladder andmicrovariant alleles; however, tri-alleles should be reamplified forverification). Microvariants will belabeled and reported as "X.Y" (where X isthe number of complete repeats and Y isthe number of basepairs of the incompleterepeat). Off-ladder will be reported as> or < the largest or smallest ladderallele, respectively. Note: thenomenclature for upload to NDIS maynecessitate a change in alleledesignation.

4.2.5.11GMID automatically flags off-scale(camera saturation) data. This data maystill be acceptable if it is limited to afew or a single peak and the overall datafor that sample is of good quality (see4.3.2.3).

4.2.5.12 Export an allele/genotypes table toExcel and save it in the run folder. Thetable will be printed for the batchrecord/file. The table should also beexported as a .cmf file for CODIS import.To create a .cmf file, the specimencategory must be assigned and the exportfields set in the CODIS Export Managerunder tools in the main menu.

4.3STR INTERPRETATION GUIDELINES

4.3.1 CONTROLS

4.3.1.1 The purpose of a REAGENT BLANK(RB) isto determine if the reagents used forDNA extraction/isolation werecontaminated with human DNA and as a


Revision 15/23/1



54/65

method for monitoring facilitydecontamination. In GeneMapperID,peaks above threshold should onlyappear in the CXR (red dye) lane,corresponding to the ILS600 size

standard. Electropherograms for theblue, green and yellow dyes should showa relatively flat baseline throughoutthe range (discounting primer signal,fluorescent 'spikes' or CXR bleed-through). If contamination (seecomment 5.2) is present in a reagentblank and does not disappear upon re-injection, results for all associatedsamples will be examined at 50 rfu forthe presence of any alleles seen in theRB. An effort shall be made todetermine the source of thecontamination, if possible. Sampledata may be deemed acceptable ifcontamination is 'isolated' to the RB.This occurrence should be documentedand the scientist's determination (andbasis for it) documented in the notepacket.

The reagent blank should be treated thesame as the least concentrated DNA

sample in terms of volume and amountamplified, injection time/amount, andanalysis threshold. Additionally, thereagent blank will be reamplified withsamples from the set if any of theamplifications conditions are moresensitive than the original.

4.3.1.2The purpose of thePOSITIVEAMPLIFICATION CONTROL(2800M DNAsupplied with the PP16 HS kit) is to

assess the amplification process,ensuring that adequate sample amplifiedsimultaneously would produce anappropriate signal. All expectedalleles (see below) must be detected,using standard parameters or all of thesamples associated with amplificationmay be deemed inconclusive. Data may


Revision 15/23/1



55/65

be deemed acceptable if all alleles arepresent (though some are below 100-rfuthreshold)ANDthe other positivecontrol (Extraction Control) appears asexpected (i.e. the problem is confined

to the 2800M sample).

LOCUS GENOTYPE LOCUS GENOTYPED3S1358 17,18 TH01 6,9.3D21S11 29,31.2 D18S51 16,18Penta E 7,14 D5S818 12,12D13S317 9,11 D7S820 8,11D16S539 9,13 CSF1PO 12,12Penta D 12,13 AMELOGENIN X,YvWA 16,19 D8S1179 14,15TPOX 11,11 FGA 20,23

4.3.1.3The purpose of theNEGATIVEAMPLIFICATION CONTROLis to determineif any human DNA contamination occurredin the process of amplification set-up(or beyond that point) and as anothermethod of monitoring facilitydecontamination. In the GeneMapperIDelectropherograms, peaks abovethreshold should only appear in the CXR

(red dye) lane, corresponding to theILS600 size standard.Electropherograms for the blue, greenand yellow dyes should show arelatively flat baseline throughout therange (discounting primer signal,fluorescent 'spikes' or CXR bleed-through). If contamination (seecomment 5.2) is present in the negativeamplification control and does notdisappear upon re-injection, results

for all of the samples associated withthat amplification will be examined forthe presence of the same peak(s). Aneffort shall be made to determine thesource of the contamination, ifpossible.


Revision 15/23/1



56/65

If extraneous peaks appear only in thiscontrol, the data for other samplesassociated with that amplification neednot be deemed inconclusive. Thisoccurrence should be documented and the

scientist's determination (and basisfor it) documented in the note packet.

4.3.1.4The purpose of an EXTRACTION CONTROLsample is primarily to assess correctgenotyping, however, it does takemeasure of all of the steps in theanalytical process from extractionthrough allele designation. Theextraction controls consist ofpreviously typed buccal samples on FTAcards prepared in batches. Anextraction control must be run on everysample plate. The reviewing scientistwill complete an Extraction ControlCheck Form (Form 212-BI) for verifyingcorrect genotype(s). A copy of thisform will be included in each databasefile. Failure of the extractioncontrol, if isolated to that sample,will not deem other samplesinconclusive.

4.3.2 RFU THRESHOLD:

4.3.2.1 For Offender database samples, aminimum of 100 rfu should be achievedfor data acceptance (the threshold maybe lowered to 50 RFU for ILS peaks asneeded). If necessary, go back in theprocess as follows: repeat injection(changing injection time; 3-10 secondsallowable range), or perform re-analysis (i.e., changing amount of

amplified product added for fragmentanalysis), or re-amplification(increase DNA template), or re-extraction.

4.3.2.2 Peaks below the analysis threshold(based on data obtained andsignal/baseline) will not be


Revision 15/23/1



57/65

interpreted but should be noted asbeing present in the notes (eg. on thetable of results).

4.3.2.3Peaks marked as off-scale in GeneMapper

ID (indicating camera saturation) willnot be interpreted if multiple peaksare affected and if it causes excessiveartifacts (e.g. split peaks, increasedstutter, pull-up, etc.) which interferewith data interpretation (see4.2.5.11). If the overall quality ofthe data is not acceptable, the samplemust be diluted, re-injected (3-10seconds), reanalyzed (decrease theamount of amplified product added) orre-amplified (decrease DNA template) asdeemed appropriate by the scientist.

4.3.2.4Multiplex amplification kits aredesigned so that heterozygous loci insingle-source samples generallydemonstrate relatively balanced peak

heights [typically 70% peak heightratio (phr)]. Some samples, althoughsingle-source, may at times demonstrategreater imbalance due to degradation,stochastic effects, primer binding site

mutations, preferential amplification,etc. Peak height ratios for these loci(


58/65

high for main peaks that are off-scaleor due to problems with spectralperformance and can be corrected bydiluting (or reamplifying less DNA) thesample and/or running a new spectral.

Loci stutter values are listed inAppendix A of the Biology QualityManual to assess potential contributionto peaks in stutter positions. N+4stutter may also be seen on occasion.

4.3.3.2Electronic or fluorescent spikesarerandom events that produce generallyspike-shaped peaks in most or all dyecolors at the same location (equivalentbp size) within a single injection.Peak heights usually vary between dyecolors for a given spike. Theseanomalies are generally notreproducible and will typically beeliminated upon reinjection. If thespike is above the analysis thresholdand falls within an allelic range thatcould interfere with either computeranalysis or scientist's analysis, thescientist will label the spike in theGeneMapperID software.

4.3.3.3Dye blobs are anomalies thattypically occur in the same approximatelocation in multiple injections and donot always disappear upon reinjection.Blobs generally look like broad orirregular peaks and may occur in asingle color or multiple colors at thesame approximate location but can varyin height. The blob should be labeledon the electropherogram (in GMID) if itfalls within a diagnostic region and is

of significant size to potentiallyinterfere with analysis.

4.3.3.4 Bleed-throughor pull-up peaks are aresult of the spectral not correctingfor all of the spectral overlap (mostcommon with the PowerPlex 16 HS kitfrom yellow into red) and may be


Revision 15/23/1



59/65

increased due to off-scale peaks.These pull-up peaks are in the samelocation (same bp size) as peaks inanother color(s) and are easilyrecognized. The presence of bleed-

through should be labeled on thecorresponding electropherogram (inGMID) if it falls within a diagnosticregion and is of significant size topotentially interfere with analysis.If excessive bleed-through occurs in acolor other than red, and is not due tooff-scale data, a new spectral may needto be performed.

4.3.3.5TaqPolymerase can catalyze theaddition of a single nucleotide(predominantly adenosine) to the 3ends of double stranded PCR product,resulting in product one base pairlonger than the actual target sequence(+A). Amplification parameters includea final extension time, so that thereaction is driven to full A addition(i.e. all product is +A). Split-peaksmay occur as a result of incomplete Aaddition and appear as a single allelerepresented by two peaks one base pair

apart (-A and +A). This can occur whenthe amount of template DNA is too great(overloaded sample). In this instance,Taq is unable to add the A nucleotideto the entire amount of productgenerated in the time allotted. Thesesamples will typically contain off-scale data as well. Split peaks can bealleviated by diluting the sample or byincubating samples at 60oC for anadditional 45 minutes, followed by

dilution, prior to reinjection. It maybe necessary to re-amplify the samplewith less template DNA.


Revision 15/23/1



60/65

4.3.4MIXTURES

4.3.4.1If, after the elimination of possiblestutter and/or bleed-thro

database analytical methods rev 13

Documents