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Current and Future tools for DNA Profiling

Daniele Podini podini@gwu.edu

Forensic Science?

Current and Future tools for DNA Profiling

Daniele Podini podini@gwu.edu

Forensic Science?

Outline

•  PCR •  Forensic Markers •  STR analysis •  Profile Frequency •  Troubleshooting •  Analytical Process •  Future Technologies

THEN THERE WAS PCR Polymerase Chain Reaction PCR

•  Polymerase Chain Reaction = molecular Xeroxing

•  “Amplify” the desired DNA fragment(s)

•  Increased sensitivity •  1988 FBI starts DNA section

Dr. Kary Mullis Eccentric Genius

1985

http://www.youtube.com/watch?v=L51UvB5za7c http://www.karymullis.com/pcr.shtml

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PolyMarker

Speed of Analysis (Technology)

Power of Discrimination

(Genetics)

Low

High

Slow Fast

RFLP Single Locus Probes

RFLP Multi-Locus Probes

ABO blood groups

DQα

D1S80

Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Limitations of DNA Testing (Past)

Multiplex STRs

Historical Perspective on DNA Typing

1985

1990

1994 1996

1998 2000

2002

1992 Capillary electrophoresis of STRs first described

First STRs developed

FSS Quadruplex

First commercial fluorescent STR

multiplexes

CODIS loci defined

Identifiler 5-dye kit and ABI 3100

PCR developed

UK National Database launched

(April 10, 1995) PowerPlex® 16 (16 loci in single amp)

2004 Y-STRs

RFLP DQA1 & PM

(dot blot) Multiplex STRs

Gill et al. (1985) Forensic application of DNA 'fingerprints‘. Nature 318:577-9

Advantages for STR Markers •  Small product sizes are generally compatible with

degraded DNA and PCR enables recovery of information from small amounts of material

•  Multiplex amplification with fluorescence detection enables high power of discrimination in a single test

•  Commercially available in an easy to use kit format

•  Uniform set of core STR loci provide capability for national and international sharing of criminal DNA profiles

Short Tandem Repeat (STR) Markers

TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAGACAGGTGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATATCATTGAAAGACAAAACAGAGATGGATGATAGATACATGCTTACAGATGCACAC

= 12 GATA repeats (“12” is all that is reported)

Target region (short tandem repeat)

7 repeats 8 repeats 9 repeats

10 repeats 11 repeats 12 repeats

13 repeats

The number of consecutive repeat units can vary between people

An accordion-like DNA sequence that occurs between genes

The FBI has selected 13 core STR loci that must be run in all DNA tests in order to provide a common currency with DNA profiles

11 repeats

12 repeats

D5S818

Short Tandem Repeats

11 repeats

12 repeats

D5S818

Short Tandem Repeats

3

11 repeats

12 repeats

D5S818

Short Tandem Repeats The ABI 310 Genetic Analyzer

ABI 310 Genetic Analyzer: Capillary Electrophoresis

Detector Window

11 12

D5S818

Short Tandem Repeats

7 8 9 10 11 12 13 14 15

D5S818

Short Tandem Repeats

11 12

D5S818

Short Tandem Repeats

D8S1179

11 9

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Scanned Gel Image Capillary Electropherogram

The polymerase chain reaction (PCR) is used to amplify STR regions and label the amplicons with

fluorescent dyes using locus-specific primers 8 repeats

10 repeats Locus 1

8 repeats

9 repeats Locus 2

Multiplex PCR (Parallel Sample Processing)

•  Compatible primers are the key to successful multiplex PCR

•  STR kits are commercially available

•  15 or more STR loci can be simultaneously amplified

Advantages of Multiplex PCR – Increases information obtained per unit time (increases power of discrimination) – Reduces labor to obtain results – Reduces template required (smaller sample consumed)

Challenges to Multiplexing  primer design to find compatible primers (no program exists)  reaction optimization is highly empirical often taking months

Statistical estimates: the product rule

0.222 x 0.222 x 2

= 0.1

Statistical estimates: the product rule

= 0.1

1 in 79,531,528,960,000,000

1 in 80 quadrillion

1 in 10 1 in 111 1 in 20

1 in 22,200

x x

1 in 100 1 in 14 1 in 81

1 in 113,400

x x

1 in 116 1 in 17 1 in 16

1 in 31,552

x x

CODIS Short Tandem Repeats

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DNA Profile Frequencies

•  The likelihood of two unrelated individuals matching at all 13 loci is less than 1 in 100 trillion (1 X 1012 - 15)

•  With 6 billion people on earth you wouldn’t expect to find two matching profiles at 13 loci

•  Only identical twins have the same profile

DNA Databasing •  DNA Databases allow for ability to search criminal

DNA profiles •  10/13/1998 FBI launched US Database

–  Combined DNA Index System (CODIS)

•  Revolutionized ability to link crime scene evidence to perpetrators

•  Databases effective >60% violent criminals are rearrested within 3yrs. for similar offense

As of February 2011: 9,404,747 offender profiles and 361,176 forensic profiles 138,700 hits assisting in more than 133,400 investigations

Biological “Artifacts” of STR Markers

•  Stutter Products •  Non-template nucleotide addition •  Microvariants •  Tri-allelic patterns •  Null alleles •  Mutations

Stutter Products •  Peaks that show up primarily one repeat less than the true allele

as a result of strand slippage during DNA synthesis

•  Stutter is less pronounced with larger repeat unit sizes (dinucleotides > tri- > tetra- > penta-)

Types of STR Repeat Units

•  Dinucleotide •  Trinucleotide •  Tetranucleotide •  Pentanucleotide •  Hexanucleotide

(CA)(CA)(CA)(CA) (GCC)(GCC)(GCC) (AATG)(AATG)(AATG) (AGAAA)(AGAAA) (AGTACA)(AGTACA)

Requires size based DNA separation to resolve different alleles from one another

Short tandem repeat (STR) = microsatellite = simple sequence repeat (SSR)

High stutter

Low stutter

YCAII

DYS448

~45%

<2%

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D21S11 D18S51

D8S1179

DNA Size (bp)

Rel

ativ

e Fl

uore

scen

ce U

nits

Stutter Product

6.3% 6.2% 5.4%

Allele

Figure 6.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

STR Alleles with Stutter Products

Taq DNA Polymerase has extended through 4 repeat units

Slipped Strand Mispairing Model

Taq has fallen off allowing the two strands to breathe apart.

Slipped Strand Mispairing Model

When the two strands re-anneal the template (bottom) strand has looped out and the extending strand aligns out-of-register by one repeat unit.

Slipped Strand Mispairing Model

The newly completed strand contains only 7 repeat units, while the template strand has the original 8 repeat units.

Slipped Strand Mispairing Model Non-Template Addition •  Taq polymerase will often add an extra nucleotide to the end of a

PCR product; most often an “A” (termed “adenylation”)

•  Dependent on 5’-end of the reverse primer; a “G” can be put at the end of a primer to promote non-template addition

•  Can be enhanced with extension soak at the end of the PCR cycle (e.g., 15-45 min @ 60 or 72 oC) – to give polymerase more time

•  Excess amounts of DNA template in the PCR reaction can result in incomplete adenylation (not enough polymerase to go around)

Best if there is NOT a mixture of “+/- A” peaks (desirable to have full adenylation to avoid split peaks)

A A

Incomplete adenylation

D8S1179

-A

+A

-A

+A

-A

+A

-A

+A

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Impact of DNA Amount into PCR

•  Too much DNA –  Off-scale peaks –  Split peaks (+/-A) –  Locus-to-locus imbalance

•  Too little DNA –  Heterozygote peak imbalance –  Allele drop-out –  Locus-to-locus imbalance

D3S1358

-A

+A

10 ng template (overloaded)

2 ng template (suggested level)

DNA Size (bp)

Rel

ativ

e Fl

uore

scen

ce (R

FUs)

100 pg template

5 pg template

DNA Size (bp)

Stochastic effect when amplifying low levels of DNA produces allele dropout

Reason that DNA Quantitation is Important Prior to Multiplex Amplification Generally 0.5 – 2.0 ng DNA template is best for STR kits

Three-Peak Patterns

D21S11

“Type 2” Balanced peak

heights

Most common in TPOX and D21S11

“Type 1” Sum of heights of two of the peaks is equal to the third

D18S51

Most common in D18S51

TPOX

Clayton et al. (2004) A genetic basis for anomalous band patterns encountered during DNA STR profiling. J Forensic Sci. 49(6):1207-1214

*

* 8

8 6

6 8

Allele 6 amplicon has “dropped out”

Imbalance in allele peak heights

Heterozygous alleles are well balanced

Impact of DNA Sequence Variation in the PCR Primer Binding Site

No mutation

Mutation at 3’-end of primer binding site

(allele dropout)

Mutation in middle of primer

binding site

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 6.9, ©Elsevier Academic Press

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA VWA

13 CODIS Core STR Loci

AMEL

AMEL

Sex-typing

Position of Forensic STR Markers on Human Chromosomes

Core

STR L

oci fo

r the

Unite

d Sta

tes

1997

D21S11

TH01

D16S539 D18S51

D8S1179

D3S1358

FGA VWA

10 SGM Plus Loci

AMEL

AMEL

Sex-typing

Position of Forensic STR Markers on Human Chromosomes

D2S1338

D19S433

SE33

SE33 (Germany)

Core

STR L

oci fo

r Eur

ope

1995 1999 2005

D10S1248

D2S441

D22S1045

European loci overlap with 8 U.S. loci

STRBase

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

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The Analytical Process

•  Extraction •  Quanitiation •  Amplification (PCR – STRs) •  Analysis (Capillary Electrophoresis)

Sources of Biological Evidence

•  Blood •  Semen •  Saliva •  Urine •  Hair •  Teeth •  Bone •  Tissue Blood stain

Only a very small amount of blood is needed to obtain a

DNA profile

Extraction 1.  Cell Lysis 2.  Prevent DNA degradation 3.  DNA purification

Cells obtained from a vaginal swab.

Perpetrator’s sperm mixed with victim’s

epithelial cells

Centrifuge

REMOVE supernatant

SDS, EDTA and proteinase K

(cell lysis buffer)

Remove a portion of the mixed stain

Incubate at 37 oC

sperm pellet

“Female Fraction”

Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Sperm obtained after differential digestion prior to DNA testing

Sperm Fraction after Differential Extraction

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Perpetrator’s sperm mixed with victim’s

epithelial cells

Centrifuge

REMOVE supernatant

SDS, EDTA and proteinase K

(cell lysis buffer)

Remove a portion of the mixed stain

SDS, EDTA and proteinase K + DTT

Incubate at 37 oC

sperm pellet

DTT lyses sperm heads

“Male Fraction” “Female Fraction” sperm pellet

Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Automation of Differential Extraction

Quantitation Slot Blot Real time PCR

Amplification (PCR of STRs) Several Kits Applied Biosystems Profiler Plus Cofiler Identifiler Plus Minifiler Promega Powerplex 16 Powerplex ESX systems

Capillary Electrophoresis Applied Biosystems has monopoly

AFTER EXTRACTION Mixed Samples •  ~99% of violent crimes are committed

by men

•  DNA Mixtures of male suspect and female victim can pose an analytical challenge, especially when the female contribution is much greater than the male = preferential amplification

•  Test for markers found only on the Y-chromosome. Only male DNA is amplified!

Y-STRs

Y-STRs •  Paternal relatives all share the same Y-STR

haplotype •  10% of Central Asian males share the same

Y-STR haplotype, thought to belong to Genghis Khan

•  Less statistical significance of inclusion •  Compared to 13 autosomal STRs •  Can increase number of Y- STR loci to increase

statistical power

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Thomas Jefferson II

Field Jefferson Peter Jefferson

President Thomas Jefferson

Eston Hemings Thomas Woodson

Different Y Haplotype

Same Y Haplotype

Jefferson Y Haplotype

Jefferson Y Haplotype

?

Figure 9.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Historical Investigation DNA Study

Modern Use of Y-STR Testing Captured December 13, 2003

Is this man really Sadaam Hussein?

Uday and Qusay Hussein

Killed July 22, 2003

Matching Y-STR Haplotype Used to

Confirm Identity

(along with allele sharing from autosomal STRs)

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Box 23.1, p. 534

Mitochondrial DNA 1 2

3 5 4

12 11 10 9

6 7 8

18 17 15 16

13 14

MtDNA Haplotype Groups: 1

2,3,6,8,11,13,15,16 4,9,10

5 7

12 14,17,18

A B

B C

C C

D B

B

B

B

B B

E

F

G

G

G

Figure 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Where are we headed Future Challenges

•  Portable all in one devices

•  Obtaining more information from a DNA sample

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Portable all in one devices

•  Device that can be operated by non lab trained personnel •  Patrol officer •  Crime Scene •  Border/Customs

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•  3 etch steps •  20 drilled holes •  1 glass-glass bonding •  1 glass-PDMS bonding

Separate

assemble

Amplify

Extract

Plexiglas interface

*Easley et al. A fully-integrated microfluidic genetic analysis system with sample in-answer out capability. PNAS. 103 (51), 2006.

DNA Purification

DNA Amplification

Injection

Separation/Detection

SPE Inlet SPE Inlet Extraction Amplification Separation Injection

+5 min for fluidic pumping, etc.

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DEVELOPMENT OF A SNP ASSAY PANEL FOR ANCESTRAL ORIGIN INFERENCE AND

INDIVIDUAL SOMATIC TRAITS

FY2009 – NIJ FORENSIC DNA R&D JANUARY 1ST 2010

NIJ grantee meeting - Chicago February 22nd 2011

Dr. Daniele Podini, Assistant Professor of Forensic Molecular Biology and Biological Sciences

Research focus:

Investigational Tool

•  Ancestry and somatic traits inference

• Use technology currently available in forensic DNA laboratories

• Could be implemented in a kit form to be used on casework as needed

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Ancestry and Phenotype Inference Investigational tool that will NOT directly identify a single suspected individual but rather will

•  Help prioritize suspect processing •  Corroborate witness testimony •  Help determine the relevance of a piece of

evidence to a crime.

The Project: •  Sample Collection •  Identify DNA markers for ancestry and somatic traits

inference. •  Develop and optimize assays to genotype the

selected markers. •  Perform statistical analyses with the data set to

determine the optimal panel(s) of markers recommended for use in the crime laboratory.

•  Develop final panels for casework application. •  Disseminate results.

Sample Collection

To date ~ 250 – Target: up to 400

The Project: •  Sample Collection •  Identify DNA markers for ancestry and somatic

traits inference. •  Develop and optimize assays to genotype the

selected SNPs. •  Perform statistical analyses with the data set to

determine the optimal panel(s) of markers recommended for use in the crime laboratory.

•  Develop final panes for casework application. •  Disseminate results.

Types of Genetic Variations •  Indels

–  Small insertion/deletions CTT------GATC CTTACGGATC

•  Small variable repeats – microsatellites –  ACGACGACGACGACGACG (6 copies) –  ACGACGACGACGACGACGACG (7 copies)

•  Single Nucleotide Polymorphisms (SNP) –  Single base pair changes GTCATTCGATT GTCAGTCGATT

http://www.science.marshall.edu/murraye/341/snps/Human%20Genetics%20MTHFR%20SNP%20Page.html

Ancestry Informative Single Nucleotide Polymorphisms

AISNPs

Single Nucleotide Polymorphisms (SNPs) that collectively give a high probability of an individual’s ancestry being from one part of the world or being derived from two or more areas of the world.

K.Kidd’s classification

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Phenotype Informative Single Nucleotide Polymorphisms

PISNPs SNPs that provide a high probability that the individual has particular phenotypes, such as a particular skin color, hair color, eye color, etc.

K.Kidd’s classification

Ancestry and Phenotype Inference SNP selection –  GWAS

–  Anthropology –  Human Pigmentation Studies

•  Skin •  Eye •  Hair

–  Other

R. A

. Stu

rm e

t al.,

Am

eric

an J

ourn

al o

f Hum

an G

enet

ics

82, 4

24 (2

008)

.

GWAS •  Genome-wide association study:

examination of variation across a genome to identify genetic associations with observable traits and/or specific populations

•  Can assay 1 million SNPs at one time (Affymetrix 6.0 array)

•  Lots of studies in the last 5 yrs

0

50

100

150

200

250

300

350

2005 2006 2007 2008 2009 2010

Number of GWAS in NHGRI Database from 2005 through December 2010

SNPs with disease associations SNPs with disease associations

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SNPs with disease associations Published  Genome-­‐Wide  Associa4ons  through  12/2009,    

658  published  GWA  at  p<5x10-­‐8  NHGRI  GWA  Catalog  www.genome.gov/GWAStudies  

Published Genome-Wide Associations through 6/2010, 904 published GWA at p<5x10-8 for 165 traits

Duffy blood group identifies phenotypes associated with two proteins that appear on the outside of red-blood cells as a receptor

http://science.uwe.ac.uk/StaffPages/na/duffy_4.gif http://www.fi.edu/learn/heart/blood/images/red-blood-cells.jpg

Example: Duffy null allele

Duffy blood group

Plays an important role in susceptibility to malaria infection (P. vivax ).

The Fy(a-b-) phenotype (rs2814778) i.e. receptor not being expressed represents an adaptation to living in malaria-endemic regions.

This is a predominant feature in African populations especially those from West Africa.

Example: Duffy null allele

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G. Tully, Forensic Science International: Genetics 1, 105 (2007).

SLC24A5 ASIP MC1R SLC45A2 OCA2

HERC 2 http://voiland.org/blog/wp-content/uploads/2008/02/blue.jpg

Pigmentation

http://www.research.uky.edu/odyssey/summer08/tan.html

Skin Pigmentation – MC1R •  In modern humans:

–  less skin pigmentation = high /varying polymorphism at MC1R –  More skin pigmentation = low polymorphism at MC1R

•  Indicates selective pressure over time favors MC1R mutations for less skin pigmentation, while need for more skin pigmentation limited variation in Africa

•  Proposed hypotheses regarding the advantages / disadvantages of pigmentation

Effects of UV Radiation on Skin

Image from Jablonski 2004

SNPs

•  An initial battery of 105 SNPs has been selected for screening:

•  46 Ancestry Informative SNPs • 59 Phenotype Informative SNPs

Modified from{http://snpsfinder.lanl.gov/

The Project: •  Sample Collection •  Identify DNA markers for ancestry and somatic

traits inference. •  Develop and optimize assays to genotype the

selected SNPs. •  Perform statistical analyses with the data set to

determine the optimal panel(s) of markers recommended for use in the crime laboratory.

•  Develop final panels for casework application. •  Disseminate results.

Single Base Extension

CTCAATGTGTAAGTTTTATTCACCTGCTAAGAAATTATTTTTTCAAAGCTAGCCTCAATATTTATTTTAAATGAGTGAACTTCAAGGCCTGAAAGAATAAACTAATACTTTACGAAATATTTTTGAAGTATAAAGAATATATTCAACATCTTTCCATGTCTCCAGATTTTAATATATGCCTTATTTTACTTTAAAAATTTTCAAATGTTTCTTTTATACACAATATGTTTCTTAGTCTGAATAACCTTTTCCTCTGCAGTATTTTTGAGCAGTGGCTCCRAAGGCACCGTCCTCTTCAAGAAGTTTATCCAGAAGCCAATGCACCCATTGGACATAACCGGGAATCCTACATGGTTCCTTTTATACCACTGTACAGAAATGGTGATTTCTTTATTTCATCCAAAGATCTGGGCTATGACTATAGCTATCTACAAGATTCAGGTAAAGTTTACTTTCTTTCAGAGGAATTGCTGAATCTAGTGTTACCAATTTATTTTGAGATAACACAAAACTTTATGCTTCGACAATGTTATTCCTGAACACTTTAAATCCTGAAAGTGCATTATAATCCTTAATTTAT

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Single Base Extension

13-Plex

Single Base Extension Summary Advantages Disadvantages •  Target small amplicons

•  Easily multiplexed

•  Increased sensitivity

•  Equipment already present in Forensic DNA Labs

•  Assay design is tedious

The Project: •  Sample Collection •  Identify DNA markers for ancestry and somatic

traits inference. •  Develop and optimize assays to genotype the

selected SNPs. •  Perform statistical analyses with the data set to

determine the optimal panel(s) of markers recommended for use in the crime laboratory.

•  Develop final panels for casework application. •  Disseminate results.

The Project: •  Sample Collection •  Identify DNA markers for ancestry and somatic

traits inference. •  Develop and optimize assays to genotype the

selected SNPs. •  Perform statistical analyses with the data set to

determine the optimal panel(s) of markers recommended for use in the crime laboratory.

•  Develop final panels for casework application. •  Disseminate results.

G/G Homozygotes Eye Color

Dark Blue Blue/Green Dark Green Hazel Light Green Grey Light Blue

Final SNP panel selection Phenotype: eye color HERC 2 - SNP rs12913832

A/A Homozygotes Eye Color

Black/Very Dark Brown

Dark Brown

Light Brown

A/G Heterozygotes Eye Color

Dark Brown Light Brown Hazel Dark Green Blue/Green Light Green Grey

174 Subjects

Homozygous G (69 ~ 40%) clear colored eyes

Homozygous A (56 ~ 32%) brown eyes

heterozygous G/A (49 ~ 28%) present both phenotypes

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Final SNP panel selection Ancestry Duffy - rs2814778 - FY (A-B-)

174 Subjects

91% of homozygous C were African American or African

83% of heterozygous C/T were African American

4% of homozygous T were African American

C/C Homozygote Ethnicity

African

African American Asian

European

C/T Heterozygote Ethnicity

African American Asian

European

Other

T/T Homozygote Ethnicity

African American Asian

European

Hispanic

Other

More work

•  Complete optimization of SBE assay •  Complete sample collection and analysis •  Continue data analysis exploring different

statistical approaches •  Complete final SNP panel selection •  Final panel development •  Optimization on forensic samples

Next Generation Sequencing Technologies

Emulsion PCR

•  Fragments, with adaptors, are PCR amplified within a water drop in oil.

•  One primer is attached to the surface of a bead. •  Used by 454, Polonator and SOLiD.

Bridge PCR

•  DNA fragments are flanked with adaptors. •  A flat surface coated with two types of primers,

corresponding to the adaptors. •  Amplification proceeds in cycles, with one end of each

bridge tethered to the surface. •  Used by Solexa.

ABI SOLID

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NGS methods

•  Sequencing millions of bases •  Low cost per base •  Entire genome can be done in a few days •  Challenges in data management •  Future applications

•  At birth sequence entire genome •  Personalized medicine •  Non human genetics •  Forensic Sciences

THANK YOU QUESTIONS? Acknowledgments: Katherine Butler, MS Michelle Peck, BS Jessica Hart, BS Dr Moses Schanfield Dr Pete Vallone NIST Dr Mike Coble NIST Dr James Landers UVA

Contact info: podini@gwu.edu, Tel. 202-242-5766