Polymerase Chain Reaction a.k.a. “How’d they get all that DNA from just a little blood?”
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Polymerase Chain Polymerase Chain Reaction Reaction a.k.a. “How’d they get a.k.a. “How’d they get all that DNA from just a all that DNA from just a little blood?” little blood?”
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Transcript of Polymerase Chain Reaction a.k.a. “How’d they get all that DNA from just a little blood?”
Polymerase Chain Polymerase Chain ReactionReaction
a.k.a. “How’d they get all that DNA a.k.a. “How’d they get all that DNA from just a little blood?”from just a little blood?”
What does PCR stand for?What does PCR stand for?
Polymerase Chain Polymerase Chain ReactionReaction
Developed by Kary Developed by Kary Mullis – Nobel PrizeMullis – Nobel Prize
What is the goal of PCR?What is the goal of PCR?
To make many copies of a small section of DNA.To make many copies of a small section of DNA.
DNA Synthesis DNA Synthesis in vitroin vitro (in a test tube). (in a test tube).
How does PCR work?How does PCR work?
Very Similar to DNA Synthesis
How does PCR work?How does PCR work?
http://www.lsic.ucla.edu/ls3/tutorials/http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.htmlgene_cloning.html
http://www.iupui.edu/~wellsctr/MMIA/htm/http://www.iupui.edu/~wellsctr/MMIA/htm/animations.htmanimations.htm
DNALCDNALC
http://wps.prenhall.com/wps/media/objects/http://wps.prenhall.com/wps/media/objects/487/498929/CDA12_2/CDA12_2a/CDA12_2a.htm487/498929/CDA12_2/CDA12_2a/CDA12_2a.htm
What is needed for a PCR What is needed for a PCR reaction?reaction?
DNA template – DNA to be copied; “target DNA template – DNA to be copied; “target sequence”sequence”
What is needed for a PCR What is needed for a PCR reaction?reaction?
PCR primers – short DNA sequences; PCR primers – short DNA sequences; complimentary to beginning and end of target complimentary to beginning and end of target sequencesequence
What is needed for a PCR What is needed for a PCR reaction?reaction?
Taq DNA Taq DNA PolymerasePolymerase
- copies DNA- copies DNA
- Can survive hot - Can survive hot temperatures needed in temperatures needed in PCR reactionPCR reaction
- From heat loving bacteria - From heat loving bacteria Thermus Thermus aquaticus.aquaticus.
What is needed for a PCR What is needed for a PCR reaction?reaction?
Nucleotides – building Nucleotides – building blocks of DNAblocks of DNA
Thermalcycler – Thermalcycler – computerized to computerized to change temperatureschange temperatures
What is needed for a PCR What is needed for a PCR reaction?reaction?
MgClMgCl22 – part of buffer, all enzymes need the – part of buffer, all enzymes need the
correct buffer (acid + salts) to workcorrect buffer (acid + salts) to work
MgCl2
What are the 3 main steps of PCR?What are the 3 main steps of PCR?
Denaturing – DNA strands separate at hydrogen bondsDenaturing – DNA strands separate at hydrogen bonds
Temperature = 95Temperature = 9500CC
What are the 3 main steps of PCR?What are the 3 main steps of PCR?
Annealing – Primers bindAnnealing – Primers bind
Temperature is dependent on primer sequenceTemperature is dependent on primer sequence
Annealing temperature = 3(G+C) + 2(A+T)Annealing temperature = 3(G+C) + 2(A+T)
# of H-bonds between nucleotides# of H-bonds between nucleotides
What are the 3 main steps of PCR?What are the 3 main steps of PCR?
Extension – DNA is copiedExtension – DNA is copied
Temperature = 72Temperature = 7200CC
PCR Reaction SummaryPCR Reaction Summary1. Denaturing
2. Annealing
3. Extension
30 cycles
How many times can target DNA How many times can target DNA be copied?be copied?
DNALCDNALC
What would someone do with What would someone do with PCR?PCR?
ForensicsForensics Identification of corpses or Identification of corpses or
body parts.body parts.
Analysis of pathogens.Analysis of pathogens.
Eliminate or link suspects Eliminate or link suspects to scene of crime through a to scene of crime through a sample (blood, human hair, sample (blood, human hair, skin, semen) left at scene skin, semen) left at scene or circumstantial links (pet or circumstantial links (pet hair, plant parts).hair, plant parts).
What would someone do with What would someone do with PCR?PCR?
Medical (genotyping)Medical (genotyping) Genetic diseases identified Genetic diseases identified
pre- or post-natally.pre- or post-natally.
Cancers identified and Cancers identified and classified.classified.
Identifying bacterial or viral Identifying bacterial or viral strains for proper strains for proper treatment.treatment.
What would someone do with What would someone do with PCR?PCR?
Food ScienceFood Science Samples can be tested for Samples can be tested for
genetic engineering traits.genetic engineering traits.
Bacterial contaminants and Bacterial contaminants and source of contamination source of contamination can be quickly identified.can be quickly identified.
Pedigrees and traits of Pedigrees and traits of valuable animal food valuable animal food stocks can be verified.stocks can be verified.
What would someone do with What would someone do with PCR?PCR?
RelationshipsRelationships Paternity.Paternity.
Evolution – developing Evolution – developing relationships between relationships between organisms or studying organisms or studying extinct species from extinct species from museum specimens.museum specimens.
Ecology – tracking Ecology – tracking organisms or defining organisms or defining biodiversity by identifying biodiversity by identifying unique species.unique species.
PCR Review Music VideoPCR Review Music Video
BioRad “When You Need to Find Out Who The Daddy Is”
BioRad “GTCA”
