Libraries

Question 1

What are genomic libraries

Answer 1

Collection of DNA fragments to represent the entire genome of an organism.

Steps-
Detergent disrupts cell membrane and DNA is extracted carefully (shears easily) and purified (phenol/chloroform removes proteins then precipitates with ethanol and Na+ ions, finally add RNase)
DNA is digested with restriction enzyme (DNA fragments are similar in size)
DNA fragments inserted into vectors with ligase
Recombinant molecules taken up by host bacteria and then screened twice (one at around 50,000 pfu per plate and the other at 500 pfu)
Transfer phages onto nylon membrane and use a labelled DNA probe to hybridise and track target sequence.
Then clones that have hybridised with probe are identified using autoradiography

Question 2

What are the quality controls of genomic library

Answer 2

Measure the purity of DNA using bioanalyser or nano spectrophotometer.
Agarose gel electrophoresis to see if the DNA fragments are of similar sizes
Using qPCR to see the efficiency of ligation

Question 3

What are the different types of vectors

Answer 3

Bacteriophages- take up to 10-20 kB of insert
Cosmids- plasmids containing cos sequence which phage loading enzyme recognises and packages them into phage head which infects bacteria to form colonies not phages (can take larger amounts of insert 30-50 kB)
BAC- bacterial artificial chromosomes are plasmids with a very low copy number origin of replication. Carry very large inserts (100-200 kB)

Question 4

What are cDNA libraries

Answer 4

Synthetic DNA that represents the transcriptome

Steps-
mRNA is isolated using oligo-dT beads (binds to polyA tail of mRNA)
Reverse transcriptase is used to create copy DNA from mRNA template
Single stranded cDNA is converted to double stranded with DNA polymerase 1 and DNA ligase
ds cDNA is ligated to lambda phage (vector) and inserted into bacteria and then screened using hybridisation or PCR techniques.

Question 5

What is the difference between genomic and cDNA libraries

Answer 5

Genomic libraries contains all DNA sequences present in genome(coding and non coding) whilst cDNA libraries only contain expressed genes.

Question 6

What is the importance of directionality in cDNA libraries and how it’s done

Answer 6

You want to preserve the orientation of the transcribed mRNA for protein expression studies or to determine protein production.

The mRNA is used to create the cDNA so the 5’-3’ orientation is important to ensure that the cDNA clones are representative of the mRNA

This is done with oligo-dT beads which initiate cDNA synthesis in the 3’ end of mRNA molecules.

Question 7

How can partial clones arise

Answer 7

these are cDNA clones that don’t contain the entire coding sequence of a gene.
This can be due to incomplete RT of mRNA, pre termination of cDNA synthesis or degradation of mRNA.

Question 8

What are the quality controls of cDNA libraries

Answer 8

Verifying the insert size to ensure vector can take it
Sequencing a subset of cDNA clones to assess the quality of library