Advantages of STR Analysis

1. Advantages of STR Analysis • Compares specific genetic markers, not whole genome. • Can be used for tiny samples or degraded samples because it makes many copies of sample.

2. STR Analysis Procedure 1. Isolate DNA 2. Polymerase Chain Reaction (PCR) Amplify (make copies) of specific loci 3.Electrophoresis - Sort fragments 4. Visualize fragments - can use various methods: Southern Blot, Stain Gel & UV Light, Fluorescent Tags

3. 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

4. STEP 2.PCR Procedure • 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

5. 5’ 3’ 5’ 3’ Starting DNA Template 3’ 5’ 3’ 5’ Separate strands (denature) Forward primer 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ Make copies (extend primers) Reverse primer Add primers (anneal) DNA Amplification with Polymerase Chain Reaction (PCR)

6. Polymerase Chain Reaction GCTATTCTGGGAGTCCAGAGTGGACGT CGATAAGACCCTCAGGTCTCACCTGCA CAC TGG A C G A T G C T

7. Original DNA target region Thermal cycle Thermal cycle Thermal cycle PCR Copies DNA Exponentially through Multiple Thermal Cycles In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created Each cycle takes less than 2 minutes from start to finish.

8. STEP 3.Electrophoresis

9. GCTCATA AATG AATG AATG AATG CTAATGCCCGTA STEP 4.Visualization of STR Alleles • 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.

10. GCTCATA AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG AATG CTAATGCCCGTA Fluorescentdye labels GCTCATA AATG CTAATGCCCGTA GCTCATA AATG AATG CTAATGCCCGTA GCTCATA AATG AATG AATG CTAATGCCCGTA GCTCATA CTAATGCCCGTA 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

11. 13 repeats 12 repeats • 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 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?

12. 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

13. To be a match - every fragment must match up.

14. Rape cases • separate male faction (sperm) DNA from female faction (epithelial) DNA • compare with DNA from blood Should this person be convicted of rape?

15. 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. How many loci were tested for each individual? 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.

16. FBI’s CODIS DNA Database Combined DNA Index System Links serial crimes & unsolved cases with repeat offenders 13 STR’s TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11