DNA Fingerprinting Using PCR

1. DNA Fingerprinting Using PCR Timothy G. Standish, Ph. D.

2. Polymorphism • Differences between humans can be attributed to two factors: • Environmental variation impacting development • Individual genes that vary between people • These genes which are variable are called polymorphic • Each person is genetically unique because of their unique set of polymorphic genes • All people are related in that the vast majority of their genes do not vary, but are identical from person to person

3. DNA Fingerprinting • DNA fingerprinting involves identification of DNA segments which vary between individuals • A set of DNA fragments polymorphic enough to provide a unique set of fragments for all individuals, can be used to identify any specific individual in a population • No single fragment will uniquely identify an individual, just as no single polymorphic genetic trait will uniquely identify a person, but a unique set of polymorphic DNA traits/fragments can serve as a reliable means of identification

4. Polymorphism In The TPA Gene • Tissue Plasminogen Activator (TPA) is a protein that functions in the cascade of reactions which break down blood clots • The gene contains 14 exons and 13 introns • Scattered within the introns are 28 Alu transposon sequences • Within intron 8 a single Alu sequence may be present, or it may be missing • Thus, as intron 8 varies with the presence or absence of Alu, it is polymorphic

5. Chromosome 8 Polymorphism The TPA Gene 1 2 3 4 5 6 7 8 9 10 11 12 13 Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7 Ex 8 Ex 9 Ex10 Ex11 Ex12 Ex13 Ex14 Intron 7 Intron 8 Intron 9 Exon 8 Exon 9 OR Intron 8 Intron 7 Intron 9 Exon 8 Exon 9 Alu Polymorphism In The TPA Gene

6. Intron 8 Alu insertion site Forward primer Forward primer Exon 8 Exon 9 Reverse primer Reverse primer 660 Base pairs OR Intron 8 Exon 8 Exon 9 Alu 960 Base pairs PCR Detection of TPA Polymorphism PCR will produce a 660 bp fragment if these primers are used 300 base pairs PCR will produce a 660 bp fragment

7. Components of a PCR Reaction • Buffer (containing Mg++) • Template DNA • 2 Primers that flank the fragment of DNA to be amplified • dNTPs • Taq DNA Polymerase (or another thermally stable DNA polymerase)

8. Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ 5’ 3’ PCR

9. Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ PCR Heat 5’ 3’

10. Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ PCR

11. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’ PCR Heat Heat 5’

12. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ PCR

13. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ PCR Heat Heat

14. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ PCR

15. 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 3’ 5’ 0 5’ T i m e 5’ 5’ 3’ 5’ 5’ 5’ Fragments of defined length 5’ 5’ 5’ 5’ 5’ PCR

16. Number 1 2 4 8 16 32 64 0 Cycles 1 2 3 4 5 6 DNA Between The Primers Doubles With Each Thermal Cycle

17. Intron 8 Alu insertion site Forward primer Forward primer Exon 8 Exon 9 Reverse primer Reverse primer 660 Base pairs OR Intron 8 Exon 8 Exon 9 Alu 960 Base pairs PCR Detection of TPA Polymorphism PCR will produce a 660 bp fragment if these primers are used 300 base pairs PCR will produce a 660 bp fragment

18. Homozygous lacking Alu Homozygous with Alu Heterozygous Gel Electrophoresis

19. The End