Polymerase chain reaction
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Polymerase Chain Reaction. a.k.a. “How’d they get all that DNA from just a little blood?”. What does PCR stand for?. Polymerase Chain Reaction Developed by Kary Mullis – Nobel Prize Received a $20,000 bonus; later sold it to Hoffman-LaRoche for $300,000,000. What is the goal of PCR?.

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Polymerase Chain Reaction

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Polymerase Chain Reaction

a.k.a. “How’d they get all that DNA from just a little blood?”


What does PCR stand for?

  • Polymerase Chain Reaction

  • Developed by Kary Mullis – Nobel Prize

  • Received a $20,000 bonus; later sold it to Hoffman-LaRoche for $300,000,000.


What is the goal of PCR?

  • To make many copies of a small section of DNA.

  • DNA Synthesis in vitro (in a test tube).


How does PCR work?

  • Very Similar to DNA Synthesis


How does PCR work?

  • http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html

  • http://www.iupui.edu/~wellsctr/MMIA/htm/animations.htm

  • DNALC

  • http://wps.prenhall.com/wps/media/objects/487/498929/CDA12_2/CDA12_2a/CDA12_2a.htm


What is needed for a PCR reaction?

  • DNA template – DNA to be copied; “target sequence”


What is needed for a PCR reaction?

  • PCR primers – short DNA sequences that bind to DNA; are complimentary to beginning and end of target sequence


What is needed for a PCR reaction?

  • Taq Polymerase

    • Polymerase copies DNA

    • From heat loving bacteria - Thermus aquaticus.

    • Can survive hot temperatures needed in PCR reaction.

    • Always copies in 5’  3’ direction


What is needed for a PCR reaction?

  • Nucleotides – building blocks of DNA

  • Thermalcycler – computerized to change temperatures


What are the 3 main steps of PCR?

  • Denaturing – DNA strands separate at hydrogen bonds

  • Temperature = 950C


What are the 3 main steps of PCR?

  • Annealing – Primers bind

  • Temperature is dependent on primer sequence

  • Annealing temperature = 3(G+C) + 2(A+T)

  • # of H-bonds between nucleotides


What are the 3 main steps of PCR?

  • Extension – DNA is copied

  • Temperature = 720C


PCR Reaction Summary

1. Denaturing

2. Annealing

3. Extension

30 cycles


How many times can target DNA be copied?

  • DNALC


What would someone do with PCR?

  • Forensics

    • Identification of corpses or body parts.

    • Analysis of pathogens.

    • Eliminate or link suspects to scene of crime through a sample (blood, human hair, skin, semen) left at scene or circumstantial links (pet hair, plant parts).


What would someone do with PCR?

  • Medical (genotyping)

    • Genetic diseases identified pre- or post-natally.

    • Cancers identified and classified.

    • Identifying bacterial or viral strains for proper treatment.


What would someone do with PCR?

  • Food Science

    • Samples can be tested for genetic engineering traits.

    • Bacterial contaminants and source of contamination can be quickly identified.

    • Pedigrees and traits of valuable animal food stocks can be verified.


What would someone do with PCR?

  • Relationships

    • Paternity.

    • Evolution – developing relationships between organisms or studying extinct species from museum specimens.

    • Ecology – tracking organisms or defining biodiversity by identifying unique species.


PCR Review Music Video

BioRad “When You Need to Find Out Who The Daddy Is”

BioRad “GTCA”


PCR Lab #1 - Alu

  • What DNA sequence are we going to copy?

    • Alu element

    • Chromosome 16

    • Does NOT code for a gene


PCR Lab #1 - Alu

  • 95% of the human genome does NOT code for proteins

    • “Junk” DNA

    • Nobody knows why we have it

    • All of it is copied during DNA synthesis and passed from one generation to the next


PCR Lab #1 - Alu

  • Alu is an example of a “jumping gene” (also known as a transposon)

    • Come from a virus infection

    • All primates share the same initial Alu sequence

    • Human chromosomes contain an estimated 1,000,000 Alu copies equaling 10% of the total genome.


PCR Lab #1 - Alu

  • Alu elements are dimorphic (meaning two forms)

    • Chromosome 16 either has this Alu element or it doesn’t

    • Since chromosomes come in pairs, a person is either ++, +-, or -- .

    • DNA sequences such as Alu are used in diagnosis of genetic disease, forensic identification, and paternity testing.


PCR Lab #1 – PTC Taster

  • What DNA sequence are we going to copy?

    • PTC taste receptor gene (produces a bitter taste)


PCR Lab #1 – PTC Taster

  • Chance discovery


PCR Lab #1 – PTC Taster

  • Being a “taster” is a dominant trait

    • Homozygous dominant – TT

    • Heterozygous – Tt

    • Homozygous recessive - tt

TASTERS

NON-TASTERS


PCR Lab #1 – PTC Taster

  • About 70% of people are tasters

    • 58% for Aboriginal people of Australia

    • 98% of Native Americans


PCR Lab #1 – PTC Taster

221 bp

  • Non-taster PCR product -

Taster PCR product -

221 bp

SNP – Single Nucleotide Polymorphism. One “letter” makes a big difference.


PCR Lab #1 – PTC Taster

  • What will be our DNA source?

    • Cheek cells

    • Rinse our mouths with a saline solution - (0.8% NaCl)


PCR Lab #1 – PTC Taster

  • Why not just use pure water to rinse our mouths?

    • What would happen to our cells in 100% water?)

Due to osmosis, cells would burst before we could collect DNA


PCR Lab #1 – PTC Taster

  • Cells are collected, allowed to settle, and then mixed with Chelex solution.

    • Negatively charged beads – removes Mg2+ which inhibits Taq polymerase.


PCR Lab #1 – PTC Taster

  • Chelex, cheek cell mixture is heated to 980C. Heat bursts cells open and cell debris is bound to Chelex beads.

  • Chelex beads and cell debris is heavy so it will settle to bottom of tube.

  • We will collect the supernatant (contains DNA) and will freeze it.

  • Set up PCR reaction on Thursday.


3’ A T C G G A C C G A C T G C A T G C C A G T T A A T A G C C A A T 5’

5’ T A G C C T G G C T G A C G T A C G G T C A A T T A T C G G T T A 3’

Denature; 950C

Taq Pol

3’ A T C G G A C C G A C T G C A T G C C A G T T A A T A G C C A A T 5’

Anneal; ~600C

Extend; 720C

5’ C T G G 3’

3’ T A G C 5’

Taq Pol

5’ T A G C C T G G C T G A C G T A C G G T C A A T T A T C G G T T A 3’


PCR Lab #2 - mtDNA

  • What DNA sequence are we going to copy?

    • mtDNA control element

    • Does NOT code for a gene


PCR Lab #2 - mtDNA

  • In which organelle is this DNA stored?

    • mitochondria

    • All DNA is NOT stored

      in nucleus!


PCR Lab #2 - mtDNA

  • How many genes are on this DNA?

    • 37 genes in 16,569bp


PCR Lab #2 - mtDNA

  • What proteins do these genes make?

    • Proteins involved in cellular respiration


PCR Lab #2 - mtDNA

  • Interesting facts regarding mtDNA

    • Always inherited from mom

    • Mutates at a constant rate


PCR Lab #2 - mtDNA


PCR Lab #2 - mtDNA

  • Compare sequences to determine genetic relationships (phylogenetic tree)


PCR Lab #2 - mtDNA

  • Interesting facts regarding mtDNA

    • Mitochondria may have been a bacterial cell at some point


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