JS 190- Introduction to STRs

I. Pre class activitiesa. Review Assignments and Schedulesb. Exam 2 moved to Weds 11 April

II. Learning Objectives

a. Short Tandem Repeats 1. Biology of STRs2. Fluorescence and Detection formats3. Stutter4. Statistics and Interpretation

b. Other markers: mtDNA and Y STRs

Short Tandem Repeats:a subgroup of tandem repeats

(Kuhl and Caskey 1993. Curr. Opin. in Genet. Dev. 3:404)

• Head to tail arrangements of sequence units (4bp),

• Common in genomes (thousands distributed)

• Polymorphic: vary in length by no. of and/or by content of repeats.

• Stably inherited on a human time scale (for most)

• Well studied b/c others are implicated in Human Diseases and therefore the subject of clinical studies.

STR Polymorphisms occur by variation in

1) The number of repeats

GAAT GAAT GAAT5' 3'

GAAT GAAT GAAT GAAT GAAT5' 3'

and/or 2) The content of the repeats

GAAT GAAT GAAT GAT GAAT5' 3'

*

Short Tandem Repeats (STRs)

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

AATG

7 repeats

8 repeats

AATG AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Primer positions define PCR product size

Fluorescent dye label

Information on 13 CODIS STRs

D18S51 

18q21.3 AGAA L18333 13 7-27 43

Locus Name

Chromosomal Location

Repeat MotifISFH format

GenBank Accession

Allele in GenBank

Allele Range

Number of Alleles Seen

CSF1PO 

5q33.3-34 TAGA X14720 12 6-16 15

FGA 

4q28 CTTT M64982 21 15-51.2 69

TH01 

11p15.5 TCAT D00269 9 3-14 20

TPOX 

2p23-pter GAAT M68651 11 6-13 10

VWA 

12p12-pter [TCTG][TCTA] M25858 18 10-24 28

D3S1358 

3p [TCTG][TCTA] Not available -- 9-20 20

D5S818 

5q21-31 AGAT G08446 11 7-16 10

D7S820 

7q11.21-22 GATA G08616 12 6-15 22

D8S1179 

8 [TCTA][TCTG] G08710 12 8-19 13

D13S317 

13q22-31 TATC G09017 13 5-15 14

D16S539 

16q24-qter GATA G07925 11 5-15 10

D21S11 21q21 Complex [TCTA][TCTG]

AP000433 29 24-38 70

520 540 560 580 600 620 640WAVELENGTH (nm)

100

80

60

40

20

0

5-FAM JOE NED ROX

Laser excitation(488, 514.5 nm)Laser excitation(488, 514.5 nm)

No

rmal

ized

Flu

ore

sce

nt

Inte

ns

ity

Fluorescent Emission Spectra for DyesFilters collect light in narrow range

Overlap is automatically calculated and subtracted using fluorescence “matrix” standards

ABI 310 Filter Set F with color contributions between dyes

Multiplex PCR• 15 Markers Can Be amplified

at once

• Sensitivities to levels less than 1 ng of DNA

• Ability to Handle Mixtures and Degraded Samples

• Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

Detection Formats

• Gel Electrophoresis

• Capillary Electrophoresis

• Microarrays (Nanogen)

• MALDITOF-MS (Sequenome)

Gel Electrophoresis System

-

Voltage

Gel

Loading well

+anode cathode

Side view Top view

Gel lanes

DNA bands

Buffer

+

-

Separation of DNA sequence length amplified products

-

+

Smallerfragments

Largerfragments

FMBIO II Detection of STR AllelesDNA samples are

loaded onto a polyacrylamide gel

STR alleles separate during electrophoresis through the gel

Sample Separation

Sample Detection (Post-Electrophoresis)

505 nm scan to detect fluorescein-labels

585 nm scan to detect TMR-labels

Example of STR test result

• 15 different STR loci may be typed on a single gel

• Scanned using a laser• and filters to assist in

detecting different colors (fluor tags)

1 432 V S

CSF1PO

TPOX

Amelogenin

THO1

vWA

Capillary Electrophoresis SystemLaser

InletBuffer

Capillary filled with polymer solution

5-20 kV- +

OutletBuffer

Sample tray

Detection window

(cathode) (anode)

Data AcquisitionSample tray moves

automatically beneath the cathode end of the capillary to deliver each sample in succession

Sample Detection

CCD Panel

ColorSeparation

Ar+ LASER (488 nm)

Fluorescence ABI Prism spectrograph

Capillary or Gel Lane

Size Separation

Labeled DNA fragments (PCR products)

Detection region

Principles of CE Sample Separation

and Detection

Results are interpreted and printed

Electropherogram:Electropherogram: ABI Prism 310 Genetic Analyzer

STR Peaks - What do They Represent?

Going back to the gel electrophoresis, large PCR fragments travel slower than small PCR fragments as electricity is applied.

Ele

ctri

cal C

urr

en

t

Smallerfragments

Largerfragments

What STR Peaks Show

By the same token, smaller PCR fragments migrate through the capillary tube faster and thus are detected before the larger (slower) PCR fragments.

Laser - Camera145

146

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153

157

145

Laser - Camera146

150

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Laser - Camera

146

150

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Laser - Camera

150

153

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Laser - Camera

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STR Peaks - What do They Represent?

NOTE: in an electropherogram,-smaller DNA fragments (bottom of traditional gel) are on the left

- the larger fragments (top of the gel) are on the right.

Larger allelicfragments

Smaller allelicfragments

STR Peaks - What do They Represent?

The area under the peak is directly proportional to the intensity of the signal.

Comparison of Gels vs CE

• Gels– Advantages

• Fewer artifacts

• Generally less expensive

• Less sensitive to ambient temperature

– Disadvantages• Not fully automated

• Need to pour and load gels

• Cannot easily reinject a sample

• CE– Advantages

• Real time detection• Better resolution of

fragments and microvariants• Fuly automated- no gel

pouring or loading• Can reinject samples• Majority of crime labs are

using CE

– Disadvantages• Generally more artifacts• More expensive• Temperature sensitive

Heterozygous versus Homozygousin SINGLE SOURCE samples

Locus 1 Locus 2 Locus 3

At each locus there are either one or two peaks. Two peaks at a locus site are called heterozygous while one peak is called homozygous.

Heterozygous Heterozygous Homozygous

STR - Mixture and StutterStutter is observed as a minor allele appearing one repeat unit smaller than the major STR allele. Some STR loci are more prone to stutter than others.

Stutter becomes an issue in putative mixed samples where a decision must be made whether a band is due to stutter or from another DNA source.

General Rule » Do stutter validation studies

STR Allele Frequencies

0

5

10

15

20

25

30

35

40

45

6 7 8 9 9.3 10

Caucasians (N=427)

Blacks (N=414)

Hispanics (N=414)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Fre

qu

ency

Probability Analysis - The Product Rule

1 in 10

1 in 20

1 in 5

If all three alleles match in two samples then1/10 x 1/20 x 1/5 = 1/1000

Allele A has a frequency in a population of 1/10.

Allele B has a frequency in a population of 1/20.

Allele C has a frequency in a population of 1/5.

FBI’s CODIS DNA DatabaseCombined DNA Index System: http://www.fbi.gov/about-us/lab/codis/ndis-statisticsUsed for linking serial crimes and unsolved cases with repeat offenders• Launched October 1998• Links all 50 states• Requires >4 RFLP markers and/or 13 core STR markers• As of February 2012

– Total number of profiles: 10,560,300– Total Forensic profiles: 417,200– Total Hits: 173,500– 166,700Investigations Aided

Why mtDNA SNPs?

• Well characterized and studied (population, evolutionary, medical and forensic studies)

• Uniparental maternal inheritance missing persons-mat. lineage ref smpls

• Relatively small size (16kb) and high copy number – good on low quantity/quality samples (hair, bone, teeth- ancient/degraded)-(Think Peterson case)

• Implicated in maternally inherited diseases : diabetes, deafness, hypertrophic cardiomyopathy and myopathy

• Analysis by DNA sequencing- more complex than STR analysis

• mtDNA - many mitotypes are only found 1X. Some use counting method for statistics. Commonly found mitotypes are as frequent as 1 in 10.

Why Y?• Applications

– Forensic investigations (98% of violent crime by men)– Biodefense- Male terrorist profiling– Genealogical and Evolutionary studies

• Advantages to Human Identity Testing– Male component isolated without differential extraction– Paternal lineages– Some cases with no spermatazoa- use Y STRs– Assess number of male donors/contributors– Same analysis as autosomal STRs

• Challenges– Y STR kits not as abundant- now 12plexes available in 2003– Some Y Haplogroups are common

– Population specific haplotying needed for new markers

Review of STRs

Intro to STRs

– Head to tail arrangements 4 bp repeat units

– Polymorphic, Common, Stably Inherited, Implicated in Diseases

– Advantages- Discrete, Small- less prone to PA, Useful on highly degraded DNA, Ability to Multiplex , Provide powerful discrimination.

– STR biological artifacts- stutter, adenylation, microvariants, null alleles, mutations

– Multiplexing STR loci provide powerful discrimination