Lecture 1 Flashcards
What are dideoxynucleotides
They are structural analogues of deoxynucleotides - lack OH group on the sugar
They can’t join on extending nucleotides and stops the chain extending any further.
They can incorporate into a DNA strand during DNA synthesis but do not permit further extension of the chain
What did sanger sequencing involve
The method exploited dideoxynucleotides. Its an example of sequencing by synthesis (replicating the strand as we sequence it)
- A DNA sample is denatured and as a new strand is synthesised using a mixture of dNTPs and ddNTPS, the new DNA strand extends an existing short DNA molecule of a known sequence called a primer.
- As each nucleotide us added to the chain, there is a chance that a terminator nucleotide will be added instead so no more bases can be added and end up with a truncated sequence
- The products are run side by side on a polyacrylamide gel to separate them according to size and the sequence can be read off the gel from the bottom upwards
What was a later development of the Sanger sequence
The use of fluorescently-labelled dideoxy bases which meant that sequencing could be performed in a single capillary tube with the bases distinguished by the colour of the fluorescent label
When was the first microbial genome published
It was the bacteriophage OX174 genome in early 1977, the image shows an electron micrograph of the virus which is a pathogen of the bacterium e.coli
What are the features of Bacteriophage ox174
It has a small genome, consists of a circular DNA molecule of 5386 base pairs.
The genome is extremely compact and had overlapping open reading frames (same section of DNA encodes two different proteins- increasing gene density)
Whats the largest single DNA molecule to have been sequenced
Phage lambda genome (48502 base pairs)
What did Fred Blattner propose
The possibility of sequencing an entire bacterial genome of 5Mb. This demonstrated considerable ambition as the total amount of DNA sequenced worldwide was only 2.3Mb
What is E.coli K-12
Originally isolated from a convalescent diphtheria patient in 1922.
It became adopted as the standard E.coli for lab studies, referred to as the workhorse of molecular biology (high rate of replication, doubling size of 20 minutes)
E.coli K-12 clone by clone sequencing
Section of the E.coli genome were clonsed into bacteriophage lambda and ordered based on informed from a genetic map.
The clones each contained about 250kb of the E.coli genome and there were a series of publications of the sequences of individual segments between 1993 and 1995
What was the first genome to be completed
A team led by Craig Venter published the 1.8Mb genome sequence of haemophilus influenzae Rd in 1995.
Instead of a clone-by-clone approach, they adopted a shot gun sequencing strategy involving the sequencing of random sections of genome followed by computational assembly of the complete chromosome
Why was H.influenzae chosen to be sequenced
Because it’s a small non-motile, gram-negative bacterium of the family Pasterurelleceae.
Its a human commensal found in the upper respiratory tract and also cause infections in the respiratory tract and middle ear.
What does Sanger-based shot gun seqeuncing involve
The bacterial chromosome is randomly fragmented through enzymatic means (e.g. using restriction enzymes) or physical methods such as sonication.
The fragments are cloned into a evtor such as a plasmid or bacteriophage and inserted into E.coli or another organism to grow many copies of each individual clone.
Individual copies are picked and stored as a library of clones which are sequenced using Sangers method
How do we assemble shot gun sequencing data
The reads are obtained from either end of each DNA fragment. They can be computationally assembled to produced long sequences
Why is a complete chromosome unlikely to be obtained directly from shot gun sequencing
There are likely to be gaps either due to regions of the genome which could not be cloned or due to repetitive sequences which could be resolved during assembly
