Human genome project Flashcards
(14 cards)
What type of sequencing was used
sanger
sanger sequencing summary
PCR to generate clones to be sequenced followed by a polymerase mediated synthesis step involving random termination of extension at each nucleotide position
purpose of random termination step
generates sequences of different lengths (by one nucleotide)
dideoxynucleotide (ddNTP)
no hydroxyl
causes termination at random
fluorescently labelled
which is added in excess, dNTP or ddNTP
dNTP
polymerase mediated extension
primer needed for polymerase to extend from
electropherogram
order of peaks gives sequence
3 key limitations of sanger sequencing
the necessity to have a clone of the DNA template (so that the levels of fluorescence emitted is adequate for detection)
the requirement that at least some sequence information is known beforehand (so that primers can bind to the template)
The short sequencing read length
reordering techniques
FISH
PCR based screening for sequence tagged sites
FISH
Extract DNA from a clone and label it a certain colour. Use this DNA as a probe in FISH. Assigns this clone to the location that lights up
STS
sequenced parts of genome even before human genome project. Design a primer based on this sequence information and screen those clones in a PCR reaction. Once you have a positive PCR reaction you can map those clones back to that location
clone contig
A series of overlapping clones whose chromosomal location had been mapped
no gaps
restriction endonuclease
small conc
produces overlapping fragments at random
overhangs
process
fragmentation>clone in vector>map using FISH or STS> fragment>fluorescent chain termination>assembly
