Polymerase chain reaction
This presentation is the property of its rightful owner.
Sponsored Links
1 / 41

Polymerase Chain Reaction PowerPoint PPT Presentation


  • 130 Views
  • Uploaded on
  • Presentation posted in: General

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?.

Download Presentation

Polymerase Chain Reaction

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Polymerase chain reaction

Polymerase Chain Reaction

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


What does pcr stand for

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

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

How does PCR work?

  • Very Similar to DNA Synthesis


How does pcr work1

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

What is needed for a PCR reaction?

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


What is needed for a pcr reaction1

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 reaction2

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 reaction3

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

What are the 3 main steps of PCR?

  • Denaturing – DNA strands separate at hydrogen bonds

  • Temperature = 950C


What are the 3 main steps of pcr1

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 pcr2

What are the 3 main steps of PCR?

  • Extension – DNA is copied

  • Temperature = 720C


Pcr reaction summary

PCR Reaction Summary

1. Denaturing

2. Annealing

3. Extension

30 cycles


How many times can target dna be copied

How many times can target DNA be copied?

  • DNALC


What would someone do with pcr

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).


Polymerase chain reaction

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.


Polymerase chain reaction

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.


Polymerase chain reaction

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

PCR Review Music Video

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

BioRad “GTCA”


Pcr lab 1 alu

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 alu1

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 alu2

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 alu3

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

PCR Lab #1 – PTC Taster

  • What DNA sequence are we going to copy?

    • PTC taste receptor gene (produces a bitter taste)


Polymerase chain reaction

PCR Lab #1 – PTC Taster

  • Chance discovery


Pcr lab 1 ptc taster1

PCR Lab #1 – PTC Taster

  • Being a “taster” is a dominant trait

    • Homozygous dominant – TT

    • Heterozygous – Tt

    • Homozygous recessive - tt

TASTERS

NON-TASTERS


Polymerase chain reaction

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 taster2

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 taster3

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 taster4

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 taster5

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 taster6

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.


Polymerase chain reaction

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’


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • What DNA sequence are we going to copy?

    • mtDNA control element

    • Does NOT code for a gene


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • In which organelle is this DNA stored?

    • mitochondria

    • All DNA is NOT stored

      in nucleus!


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • How many genes are on this DNA?

    • 37 genes in 16,569bp


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • What proteins do these genes make?

    • Proteins involved in cellular respiration


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • Interesting facts regarding mtDNA

    • Always inherited from mom

    • Mutates at a constant rate


Polymerase chain reaction

PCR Lab #2 - mtDNA


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • Compare sequences to determine genetic relationships (phylogenetic tree)


Polymerase chain reaction

PCR Lab #2 - mtDNA

  • Interesting facts regarding mtDNA

    • Mitochondria may have been a bacterial cell at some point


  • Login