Advantages of STR Analysis
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Transcript of Advantages of STR Analysis
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Compares specific genetic markers, not whole genome.
Can be used for tiny samples or degraded samples because it makes many copies of sample.
Advantages of STR Analysis
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1.1. Isolate Isolate DNA
2.2. Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) Amplify (make copies) of specific loci
3.3. Electrophoresis Electrophoresis - Sort fragments
4. 4. Visualize Visualize fragments - can use various methods: Southern Blot, Stain Gel & UV Light, Fluorescent Tags
STR Analysis Procedure
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STEP 1. Extract DNA from cell
• Break cell membrane – release nucleus
• Open nucleus - release DNA
protect DNA from enzymes
• Separate from other cell parts
• DNA must be precipitated
un-dissolved from solution
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1. Heat DNA - strands unzip (denature)
2. Cool
3. Add - Primers - “starter pieces” of DNA bind (anneal)
- to complementary sequences at beginning of specific loci
- DNA polymerase - “replicator machine” - Nucleotides (AGCT) building blocks
4. Heat to 75° C – DNA polymerization
STEP 2. PCR Procedure
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Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal) 5’3’
3’5’
Forward primer
Reverse primer
DNA Amplification with Polymerase Chain Reaction (PCR)
Separate strands
(denature)
5’
5’3’
3’
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Polymerase Chain Reaction
GCTATTCTGGGAGTCCAGAGTGGACGT
CGATAAGACCCTCAGGTCTCACCTGCA
TGG
CAC
G CG AT
TA C
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In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
PCR Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Thermal cycleThermal cycleThermal cycle
Each cycle takes less than 2 minutes from start to finish.
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STEP 3. Electrophoresis
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• Southern Blot & Autoradiography – with radioactive probes & X-ray.
• Gel may be stained and visualized with UV light after electrophoresis is complete.
• Fluorescent tags can be added to primer before running gel.
STEP 4. Visualization of STR Alleles
AATG AATG AATG AATGGCTCATA CTAATGCCCGTA
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For a specific loci there are many different alleles
Each allele varies in length due to the number of STR’s in the middle of the segment
Fluorescent dye labels
AATGAATG AATG AATG AATG
AATG AATG AATG AATG
AATG AATG AATG AATG
AATG AATG AATG AATG AATG AATG AATG AATG
AATG AATG
AATG AATG AATG
GCTCATA
GCTCATA
GCTCATA
GCTCATA
AATG AATG AATG AATGGCTCATA CTAATGCCCGTA
CTAATGCCCGTA
CTAATGCCCGTA
CTAATGCCCGTA
CTAATGCCCGTA
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• Each person has 2 alleles for a locus. M/D
• The resulting pattern of an STR sequence for a single locus has either 1 or 2 bands
• Can be homozygous or heterozygous for each locus.
• 1 band = homozygous – both fragments same length
• 2 bands = heterozygous – different fragment lengths
13 repeats
12 repeats
11 repeats
10 repeats
9 repeats
12 repeats
8 repeats
7 repeats
6 repeats
5 repeats
4 repeats
3 repeats
How many loci were tested for each individual?
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An autoradadiogram has several lanes containing DNA ladders.
Each band in these lanes contains a known length of DNA -
# of base pairs
Used for comparison to determine the length of unknown DNA bands in other lanes.
Determine # bp -
Use to determine # of STR repeats
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To be a match - every fragment must match up.
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Rape cases
Should this person be convicted of rape?
1. separate male faction (sperm) DNA from female faction (epithelial) DNA
2. compare with DNA from blood
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Paternity cases can be used to determine the parents of a child, to identify a child’s remains, in
sibling analysis and inheritance right cases.
Is each child the biological offspring of both parents?
Remember, each line represents one allele.
Each allele for each child is inherited from one of his/her parents.
How many loci were tested for
each individual?
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Combined DNA Index System
Links serial crimes & unsolved cases with repeat offenders
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
FBI’s CODIS DNA Database
13 STR’s