Lecture 17 - Organisation of the human genome 2 Flashcards
What is Sanger sequencing and what does it do
DNA clones to be sequenced are generated by standard PCR reaction
The clones are then subject to a polymerase-mediated synthesis step. Critical is the random termination of extension at each nucleotide position
The random termination results in DNA fragments of varying sizes which can be analysed to determine nucleotide sequence
(dNTPs added in excess over ddNTPs in reaction mixture)
What does dNTP stand for
Deoxynucleotide
What does ddNTP stand for
ddNTP
Describe the process of Sanger sequencing
Fluorescently label with fluorophores
Add fluorescently labelled ddNTPs to reaction mix
OR
Add unlabelled dNTPs to reaction mix
Reaction mix also contains your template that you want to sequence, primer, and DNA polymerase
What do fluorescence detection systems used to produce
Electropherogram
What is capillary electrophoresis do
allows the standard gel electrophoresis step to be bypassed
How was the human genome sequenced for the first time
Sanger sequencing yields only up to a maximum of 1000nt per reaction. This presented a major challenge for large-scale sequencing projects
Nonetheless, in 1987, the US government laid down a plan to sequence the entire Human Genome
By 1990 several other countries across the globe had signed up, and the project commenced
What are the 3 key libations 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
What was the plan for the human genome project
Construct an initial framework
Contig of large insert clones
Sequencing and final assembly
What was chromosomal DNA initially fragmented into
Large pieces and cloned into vectors known as YACs
What techniques were the clones mapped from their original chromosomal location
FISH-type experiments
PCR-based screening for STSs
what are clone contigs
A series of overlapping clones whose chromosomal location had been mapped was thus generated
How was sequencing and final assembly carried out
Random fragmentation
Ligate into vectors and make clones of each fragment in bacteria
Sanger sequence clones
Join to vector molecules and clone in bacteria
Sequence cloned fragments and then re-construct using regions of homology
Why was the whole genome shotgun sequencing stoppped
It was agreed by everyone involved in the project that the carefully laid out plan of step-by-step mapping-sequencing was the most reliable way forward
However, midway through the project a private venture (Celera Genomics) headed by Dr Craig Venter declared this cautious approach a waste of time and money
What was venters approach
Celera claimed that they could sequence the genome at a tenth of the cost being spent by the publicly funded effort and that they could do it in two years – this was almost 10 years into the public project
