Chapter 15

New Technologies and Automation

©2002 Academic Press

Anal. Biochem. 1999, 270, 148-152

Rapid Microchip CE Separation

Whitehead Institute

Fluorescein-labeled PCR Products

TMR-labeled PCR Products

Allelic ladders mixed with samples for genotyping purposes

PowerPlex™ 1.1

2.22.01.81.61.4Separation Time (minutes)

D5S818D13S317 D7S820

D16S539

vWA

TH01TP0X

CSF1P0

1111

12 1113

12

15 19

9.3/1011

11Flu

ores

cen

ce

2.4

Rapid Microchip CE Result

©2002 Academic Press

Microchip CE

PNAS (1997) 94: 10273-10278

Mass Spectrometry

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Int. J. Legal Med. (1998) 112: 45-49

Hybridization Arrays

Nucleic Acids Res. (2000) 28: e17D

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Capillary Electrophoresis

Electrophoresis. (1998) 19: 86-93

Gels

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J. Forensic Sci. (1998) 43: 1168-1180 Nucleic Acids Res. (1999) 27: e36

Capillary Arrays

http://www.cstl.nist.gov/biotech/strbase/tech.htm

Various STR Typing Technologies

STR Analysis by Hybridization on Microchips

©2002 Academic Press

STR allele containing 6 repeat units

Hybridization between probes and STR allele

Capture Probe(attached to chip) Reporter

Probe(fluorescent dye

attached)

Schematic of Nanogen STR Chip Assay

9

765

543

6

7 8

6

8

987

6

Locus D

Locus C

Locus B

Locus A

Alleles (Repeat #)

Locus A = 6,8Locus B = 3,4Locus C = 6,8Locus D = 6,6

©2002 Academic Press

Acceleration Region

Detector

Ion Extractor

Drift RegionElectric-Field Free

Pulsed Laser Beam

High-DensitySample Array

DNA Reaction Products(Size separated and drifting to the detector)

X-Y sample control

DNA separations occur in microseconds!

DNA separations occur in microseconds!

Total analysis is 5-10 seconds per sample because multiple spectra are

collected and averaged

Total analysis is 5-10 seconds per sample because multiple spectra are

collected and averaged

Schematic of Time-of-Flight Mass Spectrometry Process

©2002 Academic Press

ABI 310 Result

9.3 allele: 1071 sec

10 allele: 1073 sec

ABI 310 Result

9.3 allele: 1071 sec

10 allele: 1073 sec

Mass Spec Result

9.3 allele: 203.3 sec

10 allele: 204.8 sec

Mass Spec Result

9.3 allele: 203.3 sec

10 allele: 204.8 sec

Allele 10 COfilerTM size: 187 bp MS size:

83 bp

Allele 10 COfilerTM size: 187 bp MS size:

83 bp

Redesigned primers for mass spec workRedesigned primers for mass spec work

TH01 Alleles: CE vs. Mass Spec

©2002 Academic Press

Robot Sample Preparation

On-board thermal cycler with plate handlerPCR set-up and 310/3100 plate loading

STR results with automation

©2002 Academic Press