Genome Sequencing 4 Flashcards
How many base pairs are in the human genome?
3 billion
How many genes are there in the human genome?
20,000 to 25,000
What were the aims of the human genome project?
Determine the sequence of the bases
Identify all of the genes in the human genome
Improve tools for data analysis
What were the logistics of the human genome project?
20 labs in 6 different countries
Publicly funded
When was the draft sequence published?
2001
What is the difference between map based sequencing and shotgun sequencing?
In map based sequencing, the order of the DNA fragments is known before sequencing the fragments
In shotgun sequencing , the fragments are sequenced before they are ordered
Describe Hierarchical genome sequencing
Map based
Clone by clone
BAC by BAC vector
What is a BAC vector?
A bacterial artificial chromosome vector, a plasmid that can carry large fragments of DNA
How does Hierarchial sequencing work?
Construct a genetic map
Construction of ordered clone map
Sequencing of ordered clones
What is FISH?
Fluorescent in Situ Hybridisation
A way of measuring something on the chromosome, so you know where the genes are
Describe genome mapping
A map based on the frequencies of recombination between markers during crossing over of homologous chromosomes
The greater the frequency of recombination, the further apart the genes are thought to be
Describe shotgun sequencing
Used by celera
A faster way of sequencing genome
Whole genome fragments into short sequences which are cloned in plasmids and sequenced
Sequence are assemble using computer software
Describe next generation sequencing
Uses a single template strand that is immobilised and amplified to make a large number of identical fragments
`thousands of fragments are sequenced in parallel
Describe the method of next generation sequencing
Genome DNA is fragmented
Each fragment is isolated with a bead, which is surrounded by everything needed for PCR
Using PCR, 1,000,000 copies of each fragment is made and attached to the bead by the 5’ end
The bead is placed into a well with DNA polymerases and primers
As solution of one of the 4 nucleotides is added to wells to see if it reacts, it is then washed of and repeated with the rest of the nucleotides and then repeated again with all the nucleotides
How can you tell that the nucleotide has been reached in next generation sequencing?
If a nucleotide is complementary and added by DNA polymerase, then a pyrophosphate will be released creating a flash of light which is recorded
A colour coded graph is produced, and the height will tell you how many nucleotides have been added at a time
