To replicate DNA by amplifying DNA multiple times to create a sample large enough to analyse extensively.
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What does the sequencing reaction rely on?
DNA is antiparallel backbone strands, made of strands with a 5' end and a 3' end. Grows from 3' end. Complementary base pairing rules.
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What are the differences of PCR to normal DNA replication?
PCR can only replicate short sequences of DNA, not whole chromosomes. Requires primers, heating and cooling cycle used to separate and bind strands.
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What is the problem with heating the DNA to high temperatures?
Heat denatures DNA polymerase.
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How we do overcome this problem?
Taq polymerase is used which is derived from Thermus aquaticus that can withstand high temperatures and will not denature.
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What happens first in PCR?
The DNA sample is mixed with DNA nucleotides and Taq polymerase.
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What happens to this mixture first?
It is heated to 95 degrees to break hydrogen bonds making the samples single-stranded.
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What is then added and what are these?
Primers are added (short lengths - 10-20 bases of single-stranded DNA) which are complementary to DNA sequences.
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What happens to the mixture now?
It is cooled to 55 degrees allowing primers to bind and anneal DNA to form small sections of double-stranded DNA which DNA polymerase can bind to.
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What happens next?
Temperature raised to 72 degrees which is optimum temp for Taq polymerase. Polymerase binds to double-stranded DNA and adds free nucleotides to form new complementary strands.
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What happens once this first cycle is over?
Cycle can be repeated many times to increase amount of DNA.
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Provide a summary of PCR in 3 steps
1. Heat to 95 degrees to break hydrogen bonds and separate DNA strands. 2. Cool to 55 degrees to allow primers to bind and DNA to anneal to form double-stranded strands. 3. Heat to optimum 72 degrees for Taq polymerase to bind to double-stranded DNA
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What does Taq polymerase also do?
Adds free nucleotides to form new complementary strands.
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What is siRNA?
Small interefering RNA.
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What does siRNA have an important role in?
Genome regulation
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What is siRNA complementary to and what does this binding cause?
Complementary to mRNA. Causes destruction of mRNA.
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Other cards in this set
Card 2
Front
What does the sequencing reaction rely on?
Back
DNA is antiparallel backbone strands, made of strands with a 5' end and a 3' end. Grows from 3' end. Complementary base pairing rules.
Card 3
Front
What are the differences of PCR to normal DNA replication?
Back
Card 4
Front
What is the problem with heating the DNA to high temperatures?
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