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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 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 ©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 Ex10 Ex11 Ex12 Ex13 Ex14 Intron 7 Intron 8 Exon 8 Intron 7 Exon 8 OR Intron 8 Alu Intron 9 Exon 9 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 dNTPs Taq DNA Polymerase (or another thermally stable DNA polymerase) ©2000 Timothy G. Standish Temperature 100 Melting 94 oC PCR 50 0 T i m e 3’ 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC PCR 50 0 T i m e 3’ 5’ Heat 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC T i m e 3’ 5’ 5’ 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 3’ 5’ Heat 5’ 5’ Heat 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ ©2000 Timothy G. Standish Temperature 100 50 0 3’ 5’ 5’ Melting 94 oC PCR Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x 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 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 5’ 5’ 5’ Melting 94 oC 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ ©2000 Timothy G. Standish Temperature 100 Melting 94 oC 50 0 3’ 5’ 5’ Melting 94 oC Extension Annealing 72 oC Primers 50 oC 30x T i m e 5’ 5’ 5’ PCR 5’ 3’ Fragments of defined length 5’ 5’ 5’ 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 Homozygous Heterozygous lacking Alu with Alu ©2000 Timothy G. Standish ©2000 Timothy G. Standish
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