DNA Sequencing and analysis

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What was the Human Genome Project?
A large worldwide project to map the entire human genome
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What are terminator bases?
Modified versions of the DNA bases (adenine, guanine, cytosine and thymine) that stop DNA synthesis when added in place of an original base (e.g. G terminator added where guanine would have been and stops DNA synthesis)
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What else do terminator bases have which help with DNA sequencing?
Fluorescent tags on them to identify the end base of a fragment (each terminator base, A G C and T, have different coloured tags)
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What id the DNA for sequencing mixed with at the start of the DNA sequencing process?
A primer, an excess of nucleotides containing bases A C G T, DNA polymerase and terminator bases
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What is a thermal cycler?
A piece of equipment (used in PCR) that rapidly changes the temperature at programmed intervals in repeated cycles
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What happens when the DNA mixture is added to the thermal cycler?
The temperature is raised to 96C to separate double DNA strands, then lowered to 50C so the primers can anneal to DNA strand, then 60C so DNA polymerase can start to build up DNA strands with bases
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How are the terminator bases incorporated into the chains?
The DNA polymerase adds them to the chain just like the other bases, the addition of the termination is random and so results in lots of different lengths of chains
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What is capillary sequencing?
A technique used to separate the DNA fragments according to their size, like gel electrophoresis, but using minute capillary tubes
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Once capillary sequencing has been used, what happens?
The fluorescent markers on the terminator bases enable us to identify the end base on each fragment size and the order of the sequence of these bases (from smallest fragment to largest) shows the complementary sequence to the original DNA strand
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Why do we use computers in the final step?
Because the computers can compare all the fragments and identify overlaps and reassembles the genome to the correct order
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What is a flow cell?
A plastic slide where millions of DNA fragments are attached to and replicated in situ using PCR (forming clusters of identical DNA fragments)
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What is 'next generation sequencing'?
The terminator bases are added to the flow cell along with the DNA fragments, the terminators stop the synthesis and allow the clusters to be imaged, so the clusters are being sequenced at the same time as images are being taken
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Card 2

Front

What are terminator bases?

Back

Modified versions of the DNA bases (adenine, guanine, cytosine and thymine) that stop DNA synthesis when added in place of an original base (e.g. G terminator added where guanine would have been and stops DNA synthesis)

Card 3

Front

What else do terminator bases have which help with DNA sequencing?

Back

Preview of the front of card 3

Card 4

Front

What id the DNA for sequencing mixed with at the start of the DNA sequencing process?

Back

Preview of the front of card 4

Card 5

Front

What is a thermal cycler?

Back

Preview of the front of card 5
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