PCR
Polymerase chain reaction
Used to amplify one sample of DNA thousands of times over to create a large enough sample for extensive analysis
Key Terms
PCR
Polymerase chain reaction
Used to amplify one sample of DNA thousands of times over to create a large enough sample for extensive analysis
What is needed for PCR
Double stranded DNA - to act as a template
Free nucleotides (A,G,C,T)
DNA primers - signals to Taq polymerase where to bind and start s...
Steps in PCR
Denaturing of DNA
Annealing the DNA
Extension of DNA
Denaturing of DNA
Heat DNA saple to 95 degrees to break the H bonds between bases
Forms two seperate strands with exposed nucleotide bases
Annealing the DNA
Cool to 55 degrees to help DNA primers bind to each of the strands
Allows replication as DNA polymerase can only add to existing fragments
Synthesis of DNA in PCR
Increase the temperature to 72 (optimum for Taq polymerase)
Adds complementary bases to DNA primers building the complementary strands
...
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| Term | Definition |
|---|---|
PCR | Polymerase chain reaction Used to amplify one sample of DNA thousands of times over to create a large enough sample for extensive analysis |
What is needed for PCR | Double stranded DNA - to act as a template Free nucleotides (A,G,C,T) DNA primers - signals to Taq polymerase where to bind and start synthesising Taq polymerase - form of DNA polymerase (catalyses formation of H bonds between bases) Buffer - maintains pH |
Steps in PCR | Denaturing of DNA Annealing the DNA Extension of DNA |
Denaturing of DNA | Heat DNA saple to 95 degrees to break the H bonds between bases Forms two seperate strands with exposed nucleotide bases |
Annealing the DNA | Cool to 55 degrees to help DNA primers bind to each of the strands Allows replication as DNA polymerase can only add to existing fragments |
Synthesis of DNA in PCR | Increase the temperature to 72 (optimum for Taq polymerase) Adds complementary bases to DNA primers building the complementary strands Produces double-stranded DNA identical to target DNA |
Where des PCR occur | In a thermocycler |
Where is Taq polymerase found | Extracted from thermophilic bacteria |
Genome | The complete set of genes or genetic material present in a cell or organism |
Mitochondrial genome | Full genetic component of the mitochondrial, inherited solely from the mother |
DNA fingerprinting | Way of profiling DNA - involves using non-coding DNA (VNTRs) |
Gel electrophoresis | Technique used to separate fragments of DNA according to the length, relies on the fact that phosphates give the DNA a -ve charge |
VNTR | Variable Number Tandem Repeats Short nucleotide sequence that is repeated throughout the genome, the number of this varies at any given locus in the genome |
Applications of PCR | Investigations at crime scenes Detection of DNA Cloning of genomic DNA |
Applications of gel electrophoresis | Classification of species How related diff species are Southern blotting |
Applications of DNA profiling | Paternity tests Identify who body parts and remains belong to |
Steps in gel electrophoresis | Tray is prepared to hold gel substrate (agarose) One end of the tray contains wells for DNA samples, this area is -vely charged so the DNA travels the +ve electrode (anode) Buffers cover the DNA to prevent it drying out DNA markers can be added to help estimate sizes of fragments Shorter fragments incur less resistance so travel faster in a given time and therefore further |
How can the banding pattern be obtained after gel electrophoresis | Addn. of an fluorescent indicator that binds to DNA and is visible under UV light |
Satellite DNA | Repetitive sequences are arranged end to end, in tandem |
Mini satellite DNA | Repetitive sequences between 9-70 bp long |