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Short Tandem Repeats

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1 2 3 4 5 6. Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT. ... See discussion of Ogston sieving and Reptation in text. Short Tandem Repeats ... – PowerPoint PPT presentation

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Title: Short Tandem Repeats


1
Short Tandem Repeats
  • Dr. Jason Linville
  • University of Alabama at Birmingham
  • jglinvil_at_uab.edu

2
Short Tandem Repeats
  • DNA is found in the cells in our body.

Nucleus (Brain of the cell)
Mitochondria (more later)
3
Short Tandem Repeats
  • Eukaryotic Genomes full of repeated DNA

Repeat Unit
Repeats
Satellite DNA
100 1000s bp
???
Minisatellite DNA or Variable Number of Tandem
Repeats (VNTR)
10 100s bp
7 - 80
Microsatellite DNA or Short Tandem Repeats
2 6 bp
5 - gt40
4
Short Tandem Repeats
Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5 6
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5 6 7
  • On average, occur every 10,000 nucleotides.

5
Short Tandem Repeats
D5S818
2 alleles per locus
6
Short Tandem Repeats
Example
Locus D5S818
Alleles 7,9
Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
7
Short Tandem Repeats
  • Types of STRs

Dinucleotide, trinucleotide, tetranucleotide,
pentanucleotide, hexanucleotide.
Simple repeats identical length and sequence.
Compound repeats two or more adjacent simple
repeats.
Complex repeats repeat blocks varying in unit
length and sequence
8
13 CODIS Loci
Simple repeats with non-consensus
alleles Compound repeats with non-consensus
alleles Complex repeats
See Appendix I in text
9
Short Tandem Repeats
  • Why use tetranucleotides?

Why not larger?
  • Larger repeat units lead to larger allele size
    range difficult to multiplex
  • Larger size increases allelic dropout
  • Smaller amplicon easier amplification

10
D5S818
D13S317
D7S820
D16S539
11
Short Tandem Repeats
  • Why use tetranucleotides?

Why not smaller?
Di- and trinucleotides have an increase in
stutter formation.
8
12
11
7
repeats
12
Short Tandem Repeats
  • Which tetranucleotides to use?
  • Good Traits
  • Variation among individuals
  • Loci not linked (statistical importance)
  • Robust and reproducible
  • Low stutter and low mutation rate
  • Length 90-500 bp

13
Short Tandem Repeats
  • Which tetranucleotides to use?
  • Most common STRs originated
  • Caskey, Baylor College of Medicine
  • Forensic Science Service, England
  • Popularity largely determined by private
    companies use in multiplex kits
  • Promega Corporation (Madison, WI)
  • Applied Biosystems (Foster City, CA)

14
Short Tandem Repeats
  • Which tetranucleotides to use?
  • Selecting CODIS loci
  • CODIS Combined DNA Index System
  • FBI Laboratory STR project, April 1996 -
    November 1997
  • Chose 13 Loci to be included in the database

Textbook and STRbase (www.cstl.nist.gov/biotech/st
rbase) have detailed information on each STR loci.
15
Short Tandem Repeats
  • Commercially Available Kits
  • Promega
  • Powerplex 1.1 (or 1.2) and PowerPlex 2.1 covered
    all 13 loci (97-99)
  • PowerPlex 16, all 13 in one reaction (2000)
  • Applied Biosystems
  • AmpFlSTR Profiler Plus and AmpFlSTR Cofiler
    (97-98)
  • Identifiler (2003?)

16
Short Tandem Repeats
  • Loci Nomenclature

Intergenic DNA
3
5
5
Gene 1
Gene 2
3
3
5
5
Gene 1
Gene 2
3
Introns
17
Short Tandem Repeats
  • Loci Nomenclature

Introns STR name based on gene.
TH01 - located in the 1st intron (01) of gene for
tyrosine hydroxylase
Intergenic DNA based on chromosome
D DNA 5 chromosome 5 S single copy in
genome 818 818th locus described on Ch 5
D5S818
18
Short Tandem Repeats
  • Allele Nomenclature
  • Allele named after of repeat sequences
  • Are multiple choices which is correct?

TCAT, CATT, ATTC, TGAA, GAAT, AATG
19
Short Tandem Repeats
  • Allele Nomenclature

International Society of Forensic Haemogenetics
(ISFH) issued guidelines in 1994 and 1997
  • Intron STRs - use coding strand
  • Intergenic DNA use strand first described in
    literature of database entry (Genbank)
  • First motif that can define the repeat is used

Grandfathers.
20
DNA Monoplex
  • Monoplex means only one locus is amplified
  • For this example, we will look at D5S818.

21
Short Tandem Repeats
D5S818
2 alleles per locus
22
DNA Monoplex
11 repeats
12 repeats
23
DNA Monoplex
11 repeats
12 repeats
Million of copies of each
24
D5S818
11 12
25
Multiplex
  • In a multiplex run, more than one locus is
    amplified at one time.
  • In order to avoid overlap
  • The primer sites are moved
  • The dye is changed

26
DNA Multiplex
D5S818
11 repeats
12 repeats
D13S317
11 repeats
12 repeats
27
DNA Multiplex
D5S818
11 repeats
12 repeats
D13S317
11 repeats
12 repeats
28
D5S818
D13S317
D7S820
D16S539
29
DNA Multiplex
D5S818
11 repeats
12 repeats
vWA
11 repeats
12 repeats
30
(No Transcript)
31
Short Tandem Repeats
  • Separation of DNA
  • Amplification of STR produces a mixture of
    amplicons of different sizes
  • These amplicons must be separated in order to be
    measured.

DNA typically separated by electrophoresis.
32
Short Tandem Repeats
  • Electrophoresis of DNA
  • Phosphate groups on DNA backbone gives it a
    negative charge.
  • In an electric field, will migrate toward the
    anode ()

Can migrate through slab-gel or capillary.
33
Short Tandem Repeats
  • Separation Mechanics

See discussion of Ogston sieving and Reptation in
text.
34
Short Tandem Repeats
  • Electrophoresis of DNA

Slab-gel electrophoresis
35
Short Tandem Repeats
  • Slab Gel Electrophoresis

Agarose Gels
  • 200 nm pore size (2000 angstroms)
  • Separates large fragments
  • Unable to separate products only 4 bp apart

36
Short Tandem Repeats gt Slab Gel
  • Polyacrylamide Gels
  • 10 - 20 nm pore size (100 - 200 angstroms)
  • Crosslinking acrylamide and bisacrylamide
  • Can separate products 1 bp apart

37
Short Tandem Repeats gt Slab Gel
Polyacrylamide Gels
  • Polyacrylamide pore size affected by
    concentration of acrylamides or ratio of
    bisacrylamide to acrylamide ( 5 )
  • Polymerization is initiated by addition of
  • Ammonium persulfate (creates free radicals)
  • TEMED (stabilized)

38
Short Tandem Repeats
  • Capillary Electrophoresis
  • Samples separated through a viscous polymer
    filled capillary
  • Allows for automated injections
  • Detection measures time span from sample
    injection to detection

39
Short Tandem Repeats
  • Slab Gel v. Capillary Electrophoresis
  • CE faster run time
  • Better heat dissipation allows to be run at high
    voltage slab gels melt or smile at high
    voltage
  • CE is cleaner
  • Preparing and cleaning up gels is labor intensive
  • Slab Gels can run several samples at once
  • Only one sample per capillary at a time

40
Short Tandem Repeats
  • Electrophoresis Native v. Denaturing
  • Native Electrophoresis
  • DNA in double stranded form
  • Most agarose systems
  • Denaturing Electrophoresis
  • DNA in single stranded form
  • Allows for better separation
  • Typical in polyacrylamide gels

41
Short Tandem Repeats gt Electrophoresis
  • Denaturing Gel Electrophoresis
  • Chemicals used to keep complementary strands
    apart.
  • Formamide contains sample
  • Urea component of slab gel

Before addition to gel, samples heated to 95C to
denature strands.
42
Short Tandem Repeats
  • Allelic Ladder
  • Contains every allele
  • Run separately from samples
  • Comes with Amplification Kit
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