DNA Sequencing Flashcards
Define a genomic library?
A collection of clones which together contains copies of the entire nuclear DNA (genome) of the organism. Represent all sequences present in the nuclear genome.
How do you prepare a genomic DNA library?
1) Use pure, high molecular weight DNA free of nicks
2) Fragment genomic DNA to suitable size for ligation into vector.
3) Purify the fragments of a desired range by gel electrophoresis or sucrose gradient centrifugation.
4) Ligate purified insert DNA into lambda arms or cosmid vector to give a recombinant concatemer.
5) Package recombinant concatemer into phage particles in vitro, each phage will contain a distinct piece of genomic DNA.
6) Infect E.coli and plate. Each plaque contains many identical phage and different plaques contain different phage, each carrying a distinct fragment of genomic DNA.
What is a suitable size to fragment DNA for ligation into vector?
Require 20-25 kb for replacement vector and 40 kb for cosmid vector.
How do you fragment the DNA for ligation into vectors?
Use partial digestion so that fragments are not too small to be represented in libraries, for example frequently cutting Sau3A to get cleavage of random subset of sites and generate random collections of fragments. Or use mechanical shearing but the blunt ends produced give less efficient cloning.
How many clones are required to generate a genomic library?
Require all genomic sequences to be represented, therefore many clones required. 1-2 x10^6
How do you screen libraries?
Use a nitrocellulose filter to pick up phases from each plaque, incubate in alkaline solution to lyse phases and denature, released DNA. Then a single-stranded phage DNA will be bound to filter which can be hybridised with labelled probe and auto radiography can be used to visualise.
What are the two different approaches of ordering overlapping libraries?
Shotgun or ordered from ordered segments.
What are the current uses of genomic libraries?
Genome sequencing projects
Genetic tests
For PCR to maintain reliability for big genomes
Functional genomics
What are the possible DNA sequencing methods?
Sanger sequencing
Illuminati sequencing
Chemical (Maxam and Gilbert)
What is the Sanger method of DNA sequencing?
Enzymatic method developed in 1977 which involves extending a primer strand against a template strand. Involves four reaction terminated by ddNTPs. Introduced into the growing DNA chain in the same way as dNTPs but they lack a 3’ hydroxyl a group so no further dNTPs are able to join once they have.
Fluorescent labels used or x-Ray film
Each fragment produced is separated by polyacrylamide gel electrophoresis according to size (length)
Visualised according to tag and read of the gel.
Define a genomic clone?
A recombinant DNA containing a DNA fragment derived from the nuclear DNA (genome) of the organism.
What is the illuminati method of DNA sequencing?
Currently most widely used, easily creates DNA library
8 separate DNA libraries can be analysed per run with a typical read length of 75bp.
Approximately 18-35 Gb of data is produced per run.
Run time 4-9 days
The adapter-flanked DNA fragments for sequencing are added to a sealed glass microfabricated device, amplified (PCR) and sequences by detection of fluorescence, each dNTPs has a unique label therefore different colour.
Solid support contains adapters that hybridise complementary adapter
