04/05/12

Various names: DNA fingerprinting, DNA profiling, DNA (gene) testing I. History of DNA TypingII. Testing for identity vs. Testing to determine specific alleles or characteristics III. Many decisions must be made when testing DNAIV. Procedure I: RFLP analysis

POST-TEST 1V. Procedure II: PCR VI. PCR combination STR Test POST-TEST 2

Start Recorded Lecture Part 1 (~ 15 min.)

I. History

A. First cases utilizing DNA fingerprinting

1. Immigration -1985

2. Rape and murder

B. Immediate media blitz favoring DNA technology.

-Defense attorneys were caught off-guard.-Juries, suspects, attorneys, and judges rushed to accept the results of DNA testing. -Over 100 cases occurred before...

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DNA Typing Introduction &Procedures

1. Identifying the specific individual who is the source of a sample of DNAOR 2. Determining specific ‘characteristics’ of a sample of DNA

C. First successful challenge to exclude the DNA evidence: murder (Meaning there was DNA evidence, but the judge did not allow the jury to see it)

-The crime: police found a pregnant woman and her two-year old daughter stabbed to death.

-Blood was found on the custodian’s watch.

-Testimony by a private DNA testing company, Lifecodes Corporation stated that the blood on the watch matched the murdered woman’s.

-For the first time, the defense undertook a thorough examination of the genetic analyses and mounted the first extended effort to have DNA evidence excluded.

-They successfully argued that…1) Lifecodes did not use accepted scientific techniques. 2) Lifecodes did not follow their own procedures for interpreting their data.

-The DNA evidence was found inadmissible.

-Later that year, the custodian confessed and was found guilty.

D. Evaluation of DNA typing procedures.

1. 1989: National Association of Criminal Defense Lawyers set up a DNA Task Force; headed by Barry Scheck and Peter Neufeld.

-Critical assessment of DNA typing procedures and analysis.

2. At the same time, the National Academy of Sciences also began a more detailed look at the procedures. They issued a report that basically said...

a. The technology was accepted theoretically and for practical applications.

b. But, standards for forensic use should be determined.

3. After over 400 technical papers, over 150 court cases, 100 scientific conferences, three sets of DNA analysis guidelines, and a three year study by the National Research Council, most attorneys recommended the continuation of DNA typing.

Complete the Part 1 Recorded Lecture Self-Evaluation Questions.

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Start Recorded Lecture Part 2 (~ 41 min.)

II. There is a variety of DNA typing procedures. Different kinds of procedures provide different types of information. Labs can test for identity or test to only determine specific alleles or characteristics of certain area of the DNA

Note:Most DNA is the same from person to person. Only about 0.1- 0.5% varies between individuals.

Some DNA typing procedures DO identify the individual person who is the source of a sample of biological material.

Some DNA typing procedures DO NOT identify the individual person who is the source of a sample of biological material, but can provide some information about that person’s DNA.

No two individuals have the exact same sequence of nitrogen bases on every chromosome.

Exception:

Information about an individual gene (the exact alleles – the genotype- the person inherited for that gene) will not identify an individual.

Each person’s DNA is the same in every cell.

Exceptions:

However, these tests can provide useful information, such as if a person has an allele for genetic disorder.

An individual’s profile remains the same throughout life.

Exception:

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Questions: What are somatic cells? What is a ‘genome’?

III. Many decisions must be made when testing DNA.

A. In general, what area(s) of the DNA are tested?

When testing for identity, areas of the DNA are used that vary among individuals.

POLYMORPHISM:

DNA polymorphism:

o DNA SEQUENCE POLYMORPHISM:

o DNA LENGTH POLYMORPHISM:

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B. Specifically, what ‘Genetic Markers’ will be used?

Genetic Marker: Any segment of DNA that is known, can be identified, and tested.

1. Genetic markers that are Length Polymorphisms: Variable Number Tandem Repeats (VNTRs).

a. VNTRs: Sequences of DNA, 2 to ~80 (or more) bp, that are repeated (a few times to thousands of times).

Most VNTRs are non-coding (don’t code for proteins).Most is junk DNA.

There are many different VNTRs with different sequences repeated.

VNTRs are scattered throughout the genome on all nuclear chromosomes.

The number of repeats you have of a certain VNTR was inherited from your parents.

This is the basis for RFLP DNA testing.

b. Two categories:

1) STRs: These are short sequences of nucleotides (example: ATCG) that are repeated over and over again a number of times in tandem.

2) LTRs: These are longer sequences of nucleotides that are repeated over and over again a number of times in tandem.

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2. Examples of Genetic markers that are Sequence Polymorphisms:

a. SNPs (Snips): These are single nucleotide polymorphisms in which one nucleotide nitrogen base at a specific locus varies among individuals.

b. Sequencing larger sections of the DNA: This is the basis for Mitochondrial DNA (mtDNA) testing.

“Mitochondrial DNA (mtDNA) typing is increasingly used in human identity testing when biological evidence may be degraded or when quantities of the samples in question are limited. In humans, mtDNA is inherited strictly from the mother. Consequently, mtDNA analysis cannot discriminate between maternally related individuals (e.g., mother and daughter, brother and sister). However, this unique characteristic of mtDNA is beneficial for missing person cases when mtDNA samples can be compared to samples provided by the maternal relative of the missing person.

For humans, the mtDNA genome is approximately 16,000 bases (A, T, G, and C) in length containing a "control region" with two highly polymorphic regions. These two regions, termed Hypervariable Region I (HV1) and Hypervariable Region II (HV2), are 342 and 268 base pairs (bp) in length, respectively, and are highly variable within the human population. This sequence (the specific order of bases along a DNA strand) variability in either region provides an attractive target for forensic identification studies. Therefore, sequencing of these areas is usually performed.”

Complete the Part 2 Recorded Lecture Self-Evaluation Questions.

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Start Recorded Lecture Part 3 (~ 20 min.)

IV. Procedure I: RFLP analysis

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (Discovered by Alec Jeffreys.)

This procedure may determine identity of cells, usually with an individual test. All 46 nuclear chromosomes are typically utilized in this procedure.

Restriction Fragment Length PolymorphismThis refers to the use of restriction enzymes to cut the human DNA at restriction sites creating restriction fragments.

Restriction sites are specific sequences, usually around 6 nucleotides long.

Each person contains a specific recognition sequence in many places in the DNA.

If all 46 chromosomes are exposed to a restriction enzyme, the DNA will be cut into a number of differently sized restriction fragments.

Polymorphism= any variation between individuals.

When cut with the same restriction enzyme(s), different people will produce differently sized fragments (the number of nucleotides in each fragment will be different).

A. An example of a restriction site is the one recognized by the restriction enzyme EcoRI.

This enzyme cuts the following sequence:

GAATTCCTTAAG

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Each person's DNA is unique. Although everyone has the same restriction sites (sequences of nitrogen bases) in many areas of their DNA, they may be in different places than in other people's DNA.

Therefore, two different people, whose DNA is cut with the same restriction enzyme, will end up with restriction fragments (stretches of DNA produced by restriction enzyme cutting) of different lengths (RFLPs).

Length polymorphism:

A. VNTRs are used in the RFLP procedure

VARIABLE NUMBER TANDEM REPEATS

Complete the Part 3 Recorded Lecture Self-Evaluation Questions.

Start Recorded Lecture Part 4 (~ 17 min.)

C. Jeffries combined two discoveries: 1) Restriction enzymes that will cut DNA at specific restriction sites in the DNA and 2) VNTRs in human DNA.

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2. A restriction enzyme that cuts DNA not within the VNTR but on either side of the VNTR is selected. Because different people have VNTRs with different numbers of repeats, fragments of different lengths will be produced.

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3. When all 46 chromosomes of an individual are exposed to a restriction enzyme, the enzyme will cut the DNA anywhere the restriction site is located. The result may be tens of thousands of restriction fragments of many different sizes, from very large to very small (and many in-between). Each person will have their own unique set of restriction fragments, from the longest to the shortest.

4. Restriction sites are found in many places, not only near VNTRs. The RFLP procedure eventually will be able to ignore all other fragments and detect only the fragments containing VNTRS.

Complete the Part 4 Self-Evaluation Questions.

Start the Part 5 Recorded Lecture (~20 min.)

So the process of RFLP analysis first requires that you have a sample of DNAthat is then “cut up” (digested) in the lab using one or more restriction

enzymes.

The DNA is cut into fragments, now what? It’s still in a test tube and you can’t tell one fragment from the next.

A way of separating the different sized fragments is needed.

D. RFLPs are then separated using GEL SLABS.

Gels have microscopic pores through which the restriction fragments move when an electric current is applied. DNA has a negative charge, so the fragments travel toward the positive pole.

Smaller fragments can more easily move through the pores, so they travel from one end of the gel to the other faster than larger fragments.

Therefore, if all of the DNA fragments are placed at one end of the gel (the origin) and the electric current is applied for a certain period of time, the smaller fragments will have moved farther down the gel while the larger fragments are closer to the origin.

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The purpose of gel electrophoresis is to separate the different sized fragments. At this point the VNTR fragments cannot be distinguished from other fragments.

Sample Questions:

Does DNA have a negative or positive charge?

During electrophoresis, will DNA move toward the positive or negative pole?

Which moves faster through the gel, larger or smaller restriction fragments?

One restriction fragment (restriction fragment #1) has 2070 nucleotides. Restriction fragment #2 has 682 nucleotides. After 4 hours of electrophoresis, which restriction fragment will be closer to the positive pole? Why?

Complete the Part 5 Recorded Lecture Self-Evaluation Questions.

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Start Recorded Lecture Part 6 (~21 min.)

So now there are restriction fragments from the top of each gel lane to the bottom.

E. After gel electrophoresis, the DNA is unzipped.

F. Southern blotting: A sheet of nylon (the membrane) is placed over the gel. The single-stranded DNA is transferred to the nylon membrane. This is called Southern blotting. Southern blotting immobilizes and stabilizes the DNA.

G. How are the VNTR fragments differentiated from the other fragments?In a different part of the lab, other single-stranded pieces of DNA synthesized or obtained in the lab are used next. These small sections of DNA are called PROBES.

-Probes have a specific sequence of nucleotides. A probe can have almost any nucleotide sequence a researcher desires.

1. A single-stranded probe can bond to complementary sequences in another sample of single-stranded DNA.

2. For example, a probe might have the sequence GTTTGA. If the probe is combined with a person's single-stranded DNA, it will bind to all areas of the DNA with the sequence CAAACT.

3. Uses of probes- Probes bond to specific DNA sequences.

a. Some probes may have the sequence complementary to a specific allele of a gene. For example, if you want to test for cystic fibrosis, an allele complementary to it can be synthesized.

b. In the RFLP procedure, probes are designed with a complementary sequence to a tandem repeat and will, therefore, bond to the fragments with the repeats.

4. Probes can be radioactively labeled.

This means that a radioisotope (radioactive atom or molecule, usually 32P) is attached to the probe.

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H. Autoradiography: If a probe has hybridized (bonded) with a complementary sequence in the DNA being examined, it can be viewed after it has been exposed to x-ray film.

Complete the Part 6 Recorded Lecture Self-Evaluation Questions.

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Start the Part 7 Recorded Lecture (~10 min.)

I. Example: Paternity testing

Each individual receives one set of chromosomes from their mother and another, homologous set, from their father. Therefore, each parent contributes roughly half of an individual's restriction fragments. The mother's, baby's, and potential father(s)’s banding patterns are determined and compared.

Consider the examples on the sample autorad.

Question:

How much of their DNA do parents give to their offspring?

Restriction Digestion and Electrophoresis Animations: Go to the site below and click on play. http://www.dnalc.org/ddnalc/resources/animations.html

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Overview: Steps of RFLP

Complete the Part 7 Recorded Lecture Self-Evaluation Questions.

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DNA Typing Introduction and Procedures

POST-TEST 1

1. Some definitions are given below. Write the appropriate word for each of the definitions below.

a. Process of transferring DNA from the gel (after electrophoresis) to a nylon membrane; this immobilizes the DNA in the positions they are in at the end of electrophoresis.

b. Process of moving molecules when an electric current is applied; the DNA (with a negative charge) moves toward the positive pole; during RFLP analysis of DNA, this separates the different sized fragments because smaller fragments move faster through the gel.

c. All cells that make up the body except for sex cells; these are “body” cells.

d. A VNTR consisting of repeated segments with shorter repeated segments.

e. Areas in the DNA consisting of repeated sequences, these repeats are located adjacent to one another; usually consist of non-coding DNA; often used in DNA testing.

f. A DNA marker consisting of single nucleotide differences between individuals.

g. All of the DNA in the cell, organism, species, or population.

h. Type of DNA testing procedure relying on length polymorphisms resulting from the digestion of the DNA with restriction enzymes into different sized restriction fragments; the fragments are separated during electrophoresis and transferred to a nylon membrane during Southern blotting; a radioactive probe is applied to the DNA and autoradiography shows the positions of the fragments where probe has bound.

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i. The process of breaking the bonds between the nitrogen bases in the DNA double helix. This results in unbound nucleotides that can bond to other unbound nitrogen bases.

j. Exposing a sample of DNA that has been bound to a radioactive probe to an x-ray film. Portions of the DNA that are complementary to the probe will be viewed on the film as "bands".

k. Any variation between individuals.

l. A radioactive atom or molecule; often 32P, used to "label" molecules called probes.

m. A restriction enzyme with the restriction site GAATTC.

n. Variation between individuals in the number of nucleotides in DNA fragments produced after digestion with restriction enzymes.

o. A solidified substance composed of agar with pores throughout; used during electrophoretic procedures.

p. A single-stranded sequence of nucleotides used to target and identify specific areas of the DNA during DNA typing. They bond to specific single-stranded areas in the sample DNA being tested. In the RFLP procedure, they are radioactively labeled and may be complementary to VNTR sequences.

q. Any of several procedures used in forensics to either determine identity of cells or specific characteristics of the sample DNA.

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r. An enzyme which catalyzes chemical reactions which "cut" DNA at specific locations, producing DNA fragments of varying sizes.

s. Any area of the DNA with variations in the nitrogen base sequence.

t. The specific sequence of nitrogen bases that a specific restriction enzyme recognizes and "cuts".

u. Areas of the DNA that are known, and can be identified and tested.

2. What people have the same exact sequence of DNA nucleotides in their DNA?

3. All cells of the body have the same DNA complement (number of chromosomes) EXCEPT…

4. Explain the reasons for your answer to #3.

5. Most DNA is different from person to person.

a. True b. False

6. Approximately what percentage of the DNA is different from person to person?

7. How much of the DNA in ONE cell is required for RFLP analysis?

8. Explain your answer to #7. Why do you need this amount of the DNA?

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9. What is meant by "identity" of cells?

10. Describe the different types of VNTRs.

11. List 3 names for DNA typing.

12. DNA typing theory and techniques are not currently accepted by defense attorneys.

a. True b. False

13. RFLP: What is each letter an abbreviation for?

a. R:

b. F:

c. L:

d. P:

14. Can RFLP prove identity of cells? Explain your answer.

15. Who discovered the RFLP procedure?

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16. What is the restriction site of EcoRI and where does this restriction enzyme cut the site? Show the ends of the resulting fragments.

17. How many fragments will be produced if there are 5 restriction sites on a particular chromosome that is digested with a certain restriction enzyme?

18. Which of the following are true concerning VNTRs?a. The number of repeats in a person's VNTRs is inherited from the parents.b. Most VNTRs are junk DNA.c. Humans have only one VNTR sequence.d. VNTRs are only found on chromosomes 5 and 15.e. Each VNTR is repeated millions of times.

19. Read the following information.

Mother: -Maternal chromosome 3 = VNTR repeated 5X-Paternal chromosome 3 = VNTR repeated 8X

Father: -Maternal chromosome 3 = VNTR repeated 16X-Paternal chromosome 3 = VNTR repeated 12X

What are the possible VNTR patterns that a child of theirs might contain?

Maternal: Maternal: Paternal: Paternal:

Maternal: Maternal: Paternal: Paternal:

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20. Where are the restriction sites that are used to create length polymorphisms in the RFLP procedure?

21. Can a VNTR with restriction sites between repeats be used for RFLP analysis? Why or why not?

22. During electrophoresis… Indicate all that apply.a. gels are used with microscopic pores (tunnels) through which the DNA moves.b. DNA is positively charged, so it will move toward the negative pole.c. Larger fragments are bigger so they move faster than smaller fragments.d. After 4 hours, a fragment with 10 repeats will be closer to the positive pole than a fragment with 14 repeats.

23. The end of the gel where the DNA is loaded is called the…

24. What is meant by the size of a DNA fragment?

25. What is the purpose of electrophoresis?

26. A way of transferring the DNA to a more stable substance that would keep the fragments in their relative positions is required. What is this procedure called?

27. After electrophoresis, the DNA fragments are found in all sizes, from the smallest to the largest (the restriction sites are found in many places other than on either side of the VNTRs). How can the technician find the VNTRs among all of the fragments and pinpoint its position in the gel (which will tell the technician the size of the fragment)?

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28. The radioactive areas are "seen" by placing the membrane against an x-ray film. This procedure is called…

29. After the above procedure, the film is called an…

30. The dark areas on the film where the fragments with VNTRs are located are called…

33. Why don't scientists prefer the term DNA "fingerprinting"?

34. Describe how testing for identity is different from testing for specific characteristics or alleles of the DNA.

35. Why, theoretically, is it possible for some DNA typing procedures to definitely prove identity of cells?

36. When testing for identity, what qualities should the DNA possess?

37. Describe the immigration situation and the rape/murders that were the first cases utilizing DNA typing.

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38. Describe the first successful case where DNA typing was excluded from the court proceedings.

39. Describe major types of markers used for identity testing.

40. Describe how VNTRs are used to create length polymorphisms, in detail.

41. Describe mtDNA and the areas of the mtDNA that is tested.

42. Know the steps of RFLP in order, their purpose, and how they are performed.

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Start the Part 8 Recorded Lecture (~27 min.)

V. Procedure: PCR

A. The polymerase chain reaction (PCR) mimics DNA replication in a cell.

B. The process of PCR will make copies of a specific sequence of DNA in a test tube. PCR also is called DNA amplification.

C. Kary Mullis invented the procedure in 1984 and was awarded the Nobel Prize in 1994. This is extremely unusual. Generally the Nobel Prize is not given until after a lifetime of research, rarely for the invention of one technique. The procedure is so useful that it has been referenced in over tens of thousands of scientific publications.

D. Why is it so useful?

1. In order to detect and test, a certain number of copies of the DNA must be present. At 100% efficiency, PCR can produce over one billion copies of the DNA in a few hours. The copies can then be used for DNA typing, sequencing, etc. PCR is used to make enough DNA to detect and test. It also may even be incorporated as part of the test itself.

2. In theory, PCR is so sensitive that only one copy of the DNA is required. RFLP, on the other hand requires a much greater amount of DNA, roughly that found in a dime/quarter-sized bloodstain.

3. In addition, PCR targets a specific sequence, not many different areas of the DNA. This simplifies the study of individual genes or loci without having to wade through all three billion+ base pairs of human DNA.

4. It also makes it possible to test highly degraded DNA since only relatively small segments are needed. RFLP analysis requires fairly intact DNA.

E. Disadvantages: Is PCR too good? Because PCR is so sensitive, contamination is an ever-present concern.

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F. The specific sequences of DNA on both sides of the target sequence (the DNA being amplified) are required before PCR can be performed.

The sequences adjacent to both ends of the target DNA are called the flanking sequences.

If the flanking sequences are known, researchers can synthesize single- stranded DNA complementary to the flanking sequences for use as

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G. Basic materials and equipment:

1. Target DNA and flanking sequences (at least one copy)

2. DNA polymerase: enzyme utilized by and isolated from living cells for DNA replication

3. An ample supply of all four DNA nucleotides (A, T, C, G)

4. Large supply of two different single-stranded primers: sequences of DNA, ~20-30 nucleotides

-One primer is complementary to the flanking sequence on one side of the target DNA

-The other primer is complementary to the flanking sequence on the other side of the target DNA

-The primers are complementary to opposite strands of the target DNA

5. A machine called a thermocycler, which holds the tubes containing the above materials, is necessary to cycle the temperature at various points during the procedure.

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PCR STEPS

Cycle One

a. Heat mixture in tube to 90-95°C (194-203F) to unzip the DNA (~30 seconds to several minutes).

b. Cool mixture to 50-65°C (122-149F) to allow the single-stranded primers to bond to the unzipped flanking DNA (20 seconds to one minute).

c. Heat mixture to 72-75°C (161.6-167F) allowing the DNA polymerase to synthesize new DNA complementary to the target DNA (1-several minutes).

After this first cycle, there are now two copies.This same cycle is repeated with the two copies.

Each cycle doubles the number of copies of the target sequence.

Polymerase Chain Reaction Animation: Go to the link below and click on play. http://www.dnalc.org/ddnalc/resources/animations.html

Depending on the sequence(s) amplified, the DNA can be tested for variations between individuals using a variety of DNA typing techniques.

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Complete the Part 8 Recorded Lecture Self-Evaluation Questions.

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Start the Part 9 Recorded Lecture (~42 min.)

VI. PCR Combination Test: STR Testing (Short Tandem Repeats)

A. STRs are short sequences of DNA that are repeated a variable number of times.

For example, the entire STR 16 bp sequence of "gatagatagatagata" represents 4 copies of the repeat "gata". Tetranucleotides (repeats consisting of 4 nucleotides) are commonly amplified for use in forensics.

LTR/RFLP test. PCR/STR test.Number of base pairs per repeat

~30 to ~80. 2-5.

Size of each individual fragment (all repeats) in bp

Can be thousands…. ~100-350 bp.

Amplifiable? No. Yes.

Size of sample required

Large, cannot be highly degraded. Small, can be highly degraded.

Restriction digestion

Yes. No (PCR primers determine the fragment).

Electrophoresis Yes. Yes.

Variability Highly polymorphic. Only one individual test usually required.

Least amount of variability.Several STRs usually tested at one time for identification purposes.

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C. Using STRs in DNA identification

1. The concepts are basically the same as in RFLP, but the fragments are of shorter length – smaller number of bp in each repeat and a smaller range in repeat number.

2. DNA identification generally involves simultaneous co-amplification of several different STR sequences. These are called multiplex systems. All of the required primers, for each STR, are included in one tube for PCR.

3. After amplification, electrophoresis is performed.

-All of the amplified STRs are run together in one lane.

-Electrophoresis can be accomplished in two ways:

a. Using gel slabs (See next page.)

b. Using capillaries (p.34)

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a. Using gel slabs: The sample may then be electrophoresed on a gel, thereby separating the different sized fragments (based on number of repeats).

1) The table below lists the STRs in three different systems.

System 1: CTTv System 2: FFFL System 3: GammaSTR

Names of different STRs

CSF1PO F13A01 D16TPOX FESFPS D7THO1 F13B D13vWA LPL D5

2) Below is a picture of actual gels with samples taken from six different people.

The numbers on the right represent the number of copies of the repeat.

The alleles of a particular STR are symbolized by the number repeats.

Lanes 1-6 are the six different people.

L is a “Ladder” of DNA containing fragments with known repeat numbers (different alleles) for each of the STRs.

The STR sequences chosen here have a limited number of known repeats that do not overlap. Therefore, all bands may be seen, and the STR for each band can be determined.

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3) The link below is a database containing detailed information on each STR along with important information concerning STRs and their use.

Example: System 1: CSF1PO STR

Click here: STR Internet Database From the above site:Click on “STR Fact Sheets”.

Click on “CSF1PO”

Determine the chromosome where this STR is found:

Determine the range in the number of possible repeats:

Repeat sequence:

4) How are the bands seen?

Each amplified copy of the target DNA (an STR) remains attached to the primers.

a) The primers used during the PCR process can be labeled (or ‘tagged’) with radioactivity (a radioisotope was attached to each primer as it was made). Since each PCR product has the primers attached, each fragment will be radioactive and can be detected after Southern blotting and autoradiography. The primers are functioning as a probe, also.

b) Primers used during PCR to amplify the STRs can be ‘tagged’ with a fluorescent dye (Click here: What is Fluorescence) instead of radioactivity. Since primers are attached to each amplified copy of the target DNA, each STR (DNA fragment) will have a fluorescent tag. When the gel is run, the colors are detected with a fluorescent scanner (lasers in the scanners activate the dyes). Data collection occurs in "real time" as fluorescently labeled fragments migrate past the detector at a fixed point and viewed as they are collected. If different colored dyes are used, even overlapping STRs can be tested together.

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5) Electropherogram: A graphical representation illustrating discrete DNA fragments.

Electropherograms are produced by a scanner moving across the electrophoresed sample (or, likewise, the sample moving by the scanner). The scanner picks up the dyed DNA, and the data is plotted on a computer. The peaks are roughly analogous to bands on a gel.

Example of STR electropherogram.

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b. Using Capillary Electrophoresis instead of gel slabs

1) As the name implies, capillary electrophoresis (CE) is carried out in a microcapillary tube rather than in a gel slab.

2) A CE system provides automated gel assembly, sample loading, electrophoresis, detection, and analysis. Once samples, gel and buffer are loaded onto the instrument, the capillary is filled with gel, and the sample is loaded automatically. Up to ninety-six samples are automatically loaded and analyzed unattended.

3) Since the CE is fully automated, the chance of human error in the post-amplification handling and analysis of samples can be reduced, a significant benefit when large numbers of samples are processed.

4) CE is carried out at a much higher voltage (It’s ok for the gel to melt!). Therefore, separation of fragments from a single sample is completed in twenty minutes or less.

Click here for Capillary Electrophoresis Animation (Link is often down)

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C. Comparing STR/Gel Slabs and STR/Capillary Electrophoreis

STR/Gel Slabs STR/Capillary Electrophoreis

1. Extract the DNA from the cells.

2. PCR: Amplify the STR sites(Co-amplify all sites in multiplex systems).

Use primers “tagged” with radioactivity or a dye (fluorescent is common). This labels the sequences for viewing. A different set of ‘probes’ is not required. The primers act as probes.

Use primers “tagged” a dye (fluorescent is common). This labels the sequences for viewing. A different set of ‘probes’ is not required.

If multiplex systems are used and the primers are fluorescently tagged, use different color fluorescence for each primer set.

3. Electrophoresis separating the different sized STR fragments.

Electrophorese the samples in a gel separating the different sized fragments.

Electrophorese the samples in a microcapillary tube

The STRs are electrophoresed with “ladders” of known alleles with specific repeat numbers.

If primers were radioactive: fragments must be non-overlapping between STR systems. If fluorescently tagged

Fragments can be overlapping since different colors are used for each STR.

4. View the DNA Profile If primers were radioactive: Southern blotting & Autoradiography. If fluorescently tagged

Fluorescent primers: The samples are scanned as the fragments are electrophoresed.

5. Analysis If using radioactivity, an autorad: Fragment position is determined and compared to the ladder. If fluorescently tagged

Electropherogram: Peak color and position are compared to a known ladder.

6. Automation Can only be partially automated.

Can be almost completely automated.

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Overview: Steps PCR/STR

Complete the Part 9 Recorded Lecture Self-Evaluation Questions.

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DNA Typing Introduction and Procedures

POST-TEST 2

1. Some definitions are given below. Write the appropriate word for each of the definitions below.

a. Graphical representation illustrating DNA fragments, sequences or nucleotide bases as peaks. Each peak is roughly analogous to a band on a gel.

b. Procedure that produces copies of a sequence of DNA. The number of copies doubles with each cycle.

c. Areas of the DNA that are copied during PCR.

d. Machine that cycles the temperature during PCR.

e. Short sequences of DNA complementary to opposite strands flanking the target DNA to be amplified during PCR. They are synthesized in the lab.

f. Enzyme used by cells for DNA replication and by labs during the PCR process to synthesize new copies of the target sequence.

g. A condition of having two of the same alleles for a particular gene.

h. A condition of having two different alleles for a particular gene.

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i. What specifically is found in each of the copies produced by PCR?

j. Repeated sequences with 2-5 base pairs per repeat. Each fragment consists of ~100-350 base pairs.

k. Many STRs co-amplified and tested simultaneously are called systems.

l. What is the order of the nitrogen bases in the STR CSF1PO?

m. Electrophoretic procedure carried out in a microcapillary tube.

n. Sequences of DNA on either side of the target DNA. During PCR, synthesized DNA complementary to these sequences on opposite strands of the target sequence is used as primers.

o. DNA that has a known size or allele set. This DNA is often compared to the sample DNA being tested.

2. List the materials required for PCR.

1.

2.

3.

4.

5.

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3. Put the steps of PCR in the correct order.

-heat mixture to allow DNA polymerase to synthesize new DNA complementary tothe target DNA

-heat mixture to unzip the target DNA

-cool mixture to allow the primers to bond to the flanking DNA

4. Who invented PCR? When?

5. From one cell, one billion copies of the target sequence can be copied using PCR in approximately hours.

6. The entire genome is copied at one time using PCR.

a. True b. False

7. PCR has a disadvantage. It can be too which may

lead to .

8. Sequences on either side of the target sequence are called sequences.

9. Small DNA molecules complementary to flanking sequences that are synthesized in the lab are called…

10. The enzyme which bonds the free DNA nucleotides to the complementary target DNA during PCR is called…

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11. What is the genotype of person #5 for the vWA STR?

12. How are STR fragments “seen” in a capillary electrophoresis apparatus?

13. Describe the advantages of a capillary electrophoresis system.

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14. Describe how researchers can ‘ignore’ the portion of DNA not to be tested and only ‘see’ the areas of the DNA to be tested: RFLP and PCR/STRs.

15. Compare and contrast the procedures used to test and the characteristics of 1) RFLP LTRs and PCR/ STRs.

16. Describe, IN DETAIL, why the primers used during PCR are also used as probes when detecting STR alleles.

17. Describe how one can simultaneously amplify many STRs in one tube.

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18. Describe, in detail, how the ladder DNA is used to determine the number of repeats in the sample DNA being tested.

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