lecture 14 - dideoxy sequencing Flashcards
what type of electrophoresis should be used for different sizes
100 bp - 20,000bp: agarose gel
> 20,000 bp : pulsed field gel
10 - 4000 bases: polyacrylamide gel
what does polyacrylamide gel allow for and what makes it this way
separate chains that differ in length by just 1 nucleotide bc small pore size and 1500 volt power
how can you detect dna bands on polyacylamide gels
stain with ethidium bromide or other stains
OR
radioactively label dna and then expose to x-ray film and the beta-particles emitted by radioisotope blacken the film
in electrophoresis, what moves further
shorter segments
what are dideoxynucleotides (ddNTPs) and what do they do
have no 3’ hydroxyl group and they stop growth of dna chains bc no 3’ OH group to make phosphodiester linkage
explain manual dna sequencing
- anneal oligonucleotide primer to template
- add in dna polymerase and all 4 dNTPs but one of them is radiolabelled
- divide reaction mixture btwn 4 tubes each with different ddNTP
- each tube will end with a different nucleotide and will have all of the varying lengths
- combine polyacrylamide
electrophoresis to get the sequence of the DNA
what are some automated versions of dna sequencing
fluorescent dyes attached to ddNTPs so each of them has a different color so a fluorescence detector can identify the terminal basis and record on computer (only need one tube here)
how many bases can be determined in single sequencing reaction
800 but then they are combined together by computer programs
what are the advangtages of dideoxy sequencing
- A “long read” (~900 bases) can be obtained from each template.
- Accurate and well-tried.
- Still the best approach for sequencing regions of DNA < 50 kb
what are the disadvantages of dideoxy sequencing method
Scale-up for “massively parallel” sequencing of templates
is uneconomic (cloning fragments in plasmids is slow; as is gel electrophoresis).
