DNA Fingerprinting Using PCR Timothy G Standish Ph

DNA Fingerprinting Using PCR Timothy G. Standish, Ph. D. © 2000 Timothy G. Standish

Polymorphism Differences between humans can be attributed to two factors: 1. Environmental variation impacting development 2. 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 � © 2000 Timothy G. Standish

� DNA Fingerprinting 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 © 2000 Timothy G. Standish

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 © 2000 Timothy G. Standish

Polymorphism In The TPA Gene Chromosome 8 The TPA Gene Ex 1 1 Ex 2 2 Ex 3 3 Polymorphism 4 5 6 7 8 9 10 11 12 13 Ex 4 Ex 5 Ex 6 Ex 7 Ex 8 Ex 9 Ex 10 Ex 11 Ex 12 Ex 13 Ex 14 Intron 7 Intron 8 Exon 8 Intron 7 Exon 8 OR Intron 8 Alu Intron 9 Exon 9 © 2000 Timothy G. Standish

PCR Detection of TPA Polymorphism Intron 8 Forward primer Exon 8 Alu insertion site Reverse primer 660 Base pairs Exon 8 Forward primer PCR will produce a 660 bp fragment OR Intron 8 Alu 300 base pairs 960 Base pairs Exon 9 PCR will produce a 660 bp fragment if these primers are used Reverse primer Exon 9 © 2000 Timothy G. Standish

Components of a PCR Reaction �Buffer (containing Mg++) �Template DNA � 2 Primers that flank the fragment of DNA to be amplified �d. NTPs �Taq DNA Polymerase (or anothermally stable DNA polymerase) © 2000 Timothy G. Standish

Temperature 100 Melting 94 o. C PCR 50 0 T i m e 3’ 5’ 5’ 3’ © 2000 Timothy G. Standish

Temperature 100 Melting 94 o. C PCR 50 0 T i m e 3’ 5’ Heat 5’ 3’ © 2000 Timothy G. Standish

Temperature 100 Melting 94 o. C 50 0 PCR Melting 94 o. C Extension Annealing 72 o. C Primers 50 o. C T i m e 3’ 5’ 5’ 3’ © 2000 Timothy G. Standish

Temperature 100 Melting 94 o. C 50 0 PCR Melting 94 o. C Extension Annealing 72 o. C Primers 50 o. C 30 x T i m e 3’ 5’ Heat 5’ 5’ 3’ © 2000 Timothy G. Standish

Temperature 100 Melting 94 o. C 50 0 PCR Melting 94 o. C Extension Annealing 72 o. C Primers 50 o. C 30 x T i m e 3’ 5’ 5’ 3’ © 2000 Timothy G. Standish

Temperature 100 50 0 3’ 5’ 5’ Melting 94 o. C PCR Melting 94 o. C Extension Annealing 72 o. C Primers 50 o. C 30 x T i m e 5’ 5’ 5’ 3’ Heat 5’ 5’ Heat 5’ © 2000 Timothy G. Standish

Temperature 100 50 0 3’ 5’ 5’ PCR Melting 94 o. C Extension Annealing 72 o. C Primers 50 o. C 30 x T i m e 5’ 5’ 5’ Melting 94 o. C 5’ 3’ 5’ 5’ 5’ © 2000 Timothy G. Standish

Temperature 100 Melting 94 o. C 50 0 3’ 5’ 5’ Melting 94 o. C Extension Annealing 72 o. C Primers 50 o. C 30 x T i m e 5’ 5’ 5’ PCR 5’ 3’ Fragments of defined length 5’ 5’ 5’ © 2000 Timothy G. Standish

DNA Between The Primers Doubles With Each Thermal Cycle Number 1 2 0 1 Cycles 4 8 16 32 64 2 3 4 5 6 © 2000 Timothy G. Standish

PCR Detection of TPA Polymorphism Intron 8 Forward primer Exon 8 Alu insertion site Reverse primer 660 Base pairs Exon 8 Forward primer PCR will produce a 660 bp fragment OR Intron 8 Alu 300 base pairs 960 Base pairs Exon 9 PCR will produce a 660 bp fragment if these primers are used Reverse primer Exon 9 © 2000 Timothy G. Standish

Gel Electrophoresis Homozygous Heterozygous lacking Alu with Alu © 2000 Timothy G. Standish

© 2000 Timothy G. Standish