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14 Cards in this Set
- Front
- Back
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dideoxynucleotide chain termination sequencing |
addition of a modified deoxy called dideoxynucleotide ddntps |
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ddntps cause |
dna synthesis to terminate by containing only H and not OH at the 3' so no reaction/sequencing happens |
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4 key components to replication |
1. template 2. primer - get the reaction going 3. nucleotides 4. enzyme |
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PCR vs. PCR for sequencing |
PCR -two primers -enzyme -dntps -template dna -start with whole genome -get dsdna product - due to 2 primers PCR for sequencing -one primer -enzyme -dntps and ddntps -template dna -start with a pcr product -get ssdna product - due to one primer |
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the simplest reaction product |
primer sequencer |
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reactions are made so there are different |
options |
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dideoxy method of dna sequencing |
each band on the gel is one base longer than the previous band dds labelled with different dye excited by laser light and the colors are read automatically by a photocell and recorded in a computer G black A green T red C blue |
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when reading the dna sequence (template strand) |
it is read from the bottom to the top 3' to 5' under the primer or synthesized strand (read from gel or sequence) 5' to 3' |
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what is the purpose of the ddntps in computer automated dna sequencing |
to cause defined chain termination to label the synthesized dna |
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next- generation sequencing technologies |
many reactions at once different chemistries faster and cheaper reads the whole sequence at once |
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pyrosequencing reaction |
every time a c is incorporated, there is a flash of light one sequence added at a time so its a binary type sequence release of the pyrophosphate is used for detection |
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pyrosequencing 1 |
-dna is denatured to single strands -ssdna is attached to a bead -1 dna molecule with one bead in each reaction -initial reaction mix - dna template, primer, dna pol, 3 detection enzymes -one dntp added at a time to look for the flash of light and then removed |
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pyrosequencing 2 |
dntps may be incorporated if incorporated, p-p released enzyme uses p-p to convert p-p adp to atp another enzyme uses atp to produce light quantity of light is measured to differentiate c from cc to ccc etc the sequence is read from light emission |
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double peak |
gg or cc or aa or tt |
