The LightScanner® System

Achieve High Throughput Mutation Discovery and Genotyping

The LightScanner System

• The LightScanner System enables High Resolution Melting (Hi-Res Melting™).

– Hi-Res Melting is a post PCR technique for homogenous mutation discovery and scanning.

FAST – results in less than 5 minutes.SIMPLE – no post PCR processing.ECONOMICAL – costs significantly less than competing

technologies.

Scanning by Hi-Res Melting

• Closed-tube– dsDNA dye before PCR

– No processing, additions, or separations

– No exposure to the environment

• Rapid– 5-10 min for 96/384 samples

• Non-destructive– Downstream processing if necessary

• Minimal Maintenance– No expensive waste disposal

Essential components of Hi-Res Melting

• dsDNA binding dye• LCGreen Plus, designed specifically for detection of

heteroduplexes formed during PCR, manufactured exclusively by Idaho Technology

• High resolution instrumentation• LightScanner instrument, designed specifically for Hi-Res

melting applications

• Heteroduplex detection is a feature not shared with other dyes traditionally used in real-time PCR, such as SYBR Green I.

LCGreen® Plus Dye

0

5

10

15

20

25

30

35

40

45

50

72 74 76 78 80 82 84 86

0

5

10

15

20

25

30

35

40

45

50

72 74 76 78 80 82 84 86

Temperature (°C)

LCGreen Plus SYBR® Green I

-dF/

dT

wild type heterozygote

Heteroduplexes

0

5

10

15

20

25

30

35

40

45

50

72 74 76 78 80 82 84 86

0

5

10

15

20

25

30

35

40

45

50

72 74 76 78 80 82 84 86

Temperature (°C)

LCGreen Plus SYBR® Green I

-dF/

dT

wild type heterozygote

Heteroduplexes

Non-Saturating Dye

Saturating dye

LC Green –a “saturation” dye

LightScanner Instrument• Available in 96-

or 384- well format

• Uniformity across plate +/- 0.15°C

• 100 data points collected per °C at 0.10 °C/second

Hi-Res Melting Workflow

1. Amplify samples with LCGreen dye• 2 hrs. including PCR setup

2. Melt Samples• 5-10 minutes

3. Data Analysis• 5-10 minutes

4. Recover samples for sequencing (if necessary)• 30-60 minutes

Melting Curves

Temperature

CG

TA

Heterozygote Amplification

TwoHomoduplexes

TwoHeteroduplexes

C

A

T

G

Observed Combinationof 4 Duplexes

Melting Analysis

Melting Analysis

Melting Analysis

SNP 1 homozygote

SNP 2

SNP 1

SNP 1 + 2

Melting Analysis

SNP 1 homozygote

SNP 1 + 2

SNP 1

SNP 2

100 bp Product

C/C HomozygoteC/G HeterozygoteC/T HeterozygoteC/A Heterozygote

• Homozygotes are easily distinguished from heterozygotes

• Different heterozygotes trace unique melting paths

Detection of Homozygous Variants

Blinded Tumor Samples

Target Dosing

• Highsmith et al., Electrophoresis (1999)

• Constructed plasmids of 40%, 50%, and 60% GC content with A, C, G, or T at one position

• PCR primers on each side spaced 50 bp apart

Use of a DNA “toolbox” as a model system for mutation scanning

X

Reed and WittwerClin. Chem. 2004,50(10): 1748-54

Dependence on Product Size

•N=1248, each instrument•Three different targets•All possible SNPs and WTs

Clin Chem. 2004;50:1748-54.

Sensitivity and Specificity

Benefits of Hi-Res Melting Platform

• What about Hi-Res Melting on real-time PCR instruments?– Commercially available real-time PCR instruments lack the

precise temperature control, the data density acquisition and the analysis software required for successful implementation of the method.Instrument Feature LightScanner Real-time

platforms

Resolution High Low

Analysis software specific for Hi-Res Melting

Yes No

Fluorescent Compatibility Yes Some, not all compatible with LCGreen dye

Time to result 5 minutes >30 minutes

Normalized melting curves of 110-bp amplicon in the presence of LCGreen Plus. Figures demonstrate the advantage of Hi-Res Melting Platform. True Hi-Res Melting profiles enable discrimination of wild type (AA) in green, heterozygotes (AT) in blue and homozygous mutants (TT) in red.

• The power of DNA melting analysis depends directly on the resolution of the melting instrument.

• Precision of the melt curves enables identification of different sample genotypes.

Benefits of Hi-Res Melting Platform

HR-1 Instrument Commercial Real-Time PCR Instrument

HR-1 Instrument Commercial Real-Time PCR Instrument

Assumptions:DHPLC at 2 temperaturesSequencing in 1 direction using 48 capillary analyser & software detection

Approximate time required to process 96 samples

Appro

xim

ate

tota

l ela

pse

d

tim

e

Data Taken from Dr C Taylor: Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, UK

Sample Throughput

DHPLC, FSSCP and sequencing costs based on actual costs incurred for samples processed 2002-2003. Hi-Res Melting costs based on actual consumables cost and projected use.

Data Taken from Dr C Taylor: Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, UK

Project Cost

Eliminate 99% of Sequencing?

Scanning of PCR fragments for variants

DNA sequencing

~1% Not identified

Unlabeled probe genotyping of

known variants

Variant genotyped

~ 9% Identified

~ 90% Normal

~10% Abnormal

Wild type

Conclusions

• The LightScanner System includes all components necessary for successful Hi-Res Melting Applications– LCGreen Plus Dye– LightScanner Master Mix– LightScanner Instrument

• Software available for assay design, mutation discovery and genotyping

– Assays Online Protocols

• Save Time and Money• Achieve High Throughput Mutation Discovery and Gene

Scanning

Q & A

For more information about the LightScanner

System please visit www.idahotech.com