Foundations of Biology

Foundations of Biology

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

Recently Viewed Presentations

  • Working Group 6 - Federal Communications Commission | The ...

    Working Group 6 - Federal Communications Commission | The ...

    Background. Recognizing the advantages of building security in to hardware and software (rather than retrofitting), FCC has urged industry to examine security by design practices for core network equipment
  • PowerPoint Sunusu

    PowerPoint Sunusu

    The obturator sign. Pain on passive internal rotation of the flexed thigh.
  • Part II: Preparation/Process - Professor Mark J. Grossman

    Part II: Preparation/Process - Professor Mark J. Grossman

    Receiver's Bias: Peer Groups and Media. Peer pressure influences the way messages are perceived. Peer groups influence attitudes and actions. Media is a powerful agenda setter - tells us what issues are important
  • A Midsummer night's dream

    A Midsummer night's dream

    A Midsummer night's dream. Today: Understanding Shakespeare. Understanding the interaction of the characters and how this advances the plot. Identify topics from the story and compare/contrast them to modern day media
  • What's new in TypeScript

    What's new in TypeScript

    Type system. Gradual, Structural, Generic. Extensive type inference. Control flow based type analysis. Union, intersection, and tuple types. Index, lookup, and mapped ...
  • Developmental Math DSMA0300  Pre-algebra DSMA0301  Beginning Algebra DSMA0303

    Developmental Math DSMA0300 Pre-algebra DSMA0301 Beginning Algebra DSMA0303

    DSMA0300 Perform basic operations of addition, subtraction, multiplication and division of whole numbers, fractions, decimals and signed numbers, (on algebraic fractions and transfer this knowledge to the solution of application problems. DSMA0300 Solve equations. (This is the number one objective...
  • -nt -nd -nk - Education Extras

    -nt -nd -nk - Education Extras

    geo therm scope meter logy telescope thermal perimeter mythology geography thermometer barometer periscope geothermal geology thermostat millimeter horoscope microscope speedometer geometry stethoscope geode thermos ecology diameter zoology kilometer etymology Derivational - Sort 29 (geo, therm, scope, meter, logy) Greek Roots
  • Basic Statistics for the Behavioral Sciences

    Basic Statistics for the Behavioral Sciences

    Animatism Animatism is a belief that the world is animated by impersonal supernatural powers. Unlike animism, the form of power is ambiguous it does not take a particular shape, emotion, etc. Often found in similar cultures where animism is found.