Title: Short Tandem Repeats
1Short Tandem Repeats
- Dr. Jason Linville
- University of Alabama at Birmingham
- jglinvil_at_uab.edu
2Short Tandem Repeats
- DNA is found in the cells in our body.
Nucleus (Brain of the cell)
Mitochondria (more later)
3Short 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
4Short 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.
5Short Tandem Repeats
D5S818
2 alleles per locus
6Short Tandem Repeats
Example
Locus D5S818
Alleles 7,9
Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
7Short Tandem Repeats
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
813 CODIS Loci
Simple repeats with non-consensus
alleles Compound repeats with non-consensus
alleles Complex repeats
See Appendix I in text
9Short 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
10D5S818
D13S317
D7S820
D16S539
11Short Tandem Repeats
- Why use tetranucleotides?
Why not smaller?
Di- and trinucleotides have an increase in
stutter formation.
8
12
11
7
repeats
12Short 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
13Short 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)
14Short 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.
15Short 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?)
16Short Tandem Repeats
Intergenic DNA
3
5
5
Gene 1
Gene 2
3
3
5
5
Gene 1
Gene 2
3
Introns
17Short Tandem Repeats
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
18Short Tandem Repeats
- Allele named after of repeat sequences
- Are multiple choices which is correct?
TCAT, CATT, ATTC, TGAA, GAAT, AATG
19Short Tandem Repeats
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.
20DNA Monoplex
- Monoplex means only one locus is amplified
- For this example, we will look at D5S818.
21Short Tandem Repeats
D5S818
2 alleles per locus
22DNA Monoplex
11 repeats
12 repeats
23DNA Monoplex
11 repeats
12 repeats
Million of copies of each
24D5S818
11 12
25Multiplex
- 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
26DNA Multiplex
D5S818
11 repeats
12 repeats
D13S317
11 repeats
12 repeats
27DNA Multiplex
D5S818
11 repeats
12 repeats
D13S317
11 repeats
12 repeats
28D5S818
D13S317
D7S820
D16S539
29DNA Multiplex
D5S818
11 repeats
12 repeats
vWA
11 repeats
12 repeats
30(No Transcript)
31Short Tandem Repeats
- 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.
32Short Tandem Repeats
- 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.
33Short Tandem Repeats
See discussion of Ogston sieving and Reptation in
text.
34Short Tandem Repeats
Slab-gel electrophoresis
35Short Tandem Repeats
Agarose Gels
- 200 nm pore size (2000 angstroms)
- Separates large fragments
- Unable to separate products only 4 bp apart
36Short Tandem Repeats gt Slab Gel
- 10 - 20 nm pore size (100 - 200 angstroms)
- Crosslinking acrylamide and bisacrylamide
- Can separate products 1 bp apart
37Short 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)
38Short 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
39Short 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
40Short 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
41Short 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.
42Short Tandem Repeats
- Contains every allele
- Run separately from samples
- Comes with Amplification Kit