Gene Libraries Flashcards
What is a DNA library?
Chunks of genomes archived in a library
What is a partial restriction digest?
Not a full digest
There are random overlapping fragments of DNA so that pieces can be put back together like a puzzle
What are the requirements of a cDNA Library?
Need to isolate the mRNA
Need to form cDNA using reverse transcriptase
Need cloning of the DNA
What are the steps in cDNA library formation?
1) Cell lysis
2) RNA extraction
3) Purification of Total RNA, poly mRNAs and small RNAs
4) Reverse transcription of mRNA with primers of random hexamers, oligos
5) RNase H digests RNA strand in an RNA-DNA hybrid
6) 3’ hairpin loop self-primes ss cDNA
7) DNA polymerase copies the primed cDNA
8) Nuclease cleaves the ssDNA loop
9) ds cDNA produced which can be then cloned into a large vector
What is the advantage of cDNA library over Genomic library?
No fragmentation
Only expressed genes from the source
Shorter than whole genes
Fewer clones needed= so a larger vector choice
cDNA libraries are only useful if they can be screened for the sequence of interest. What are some ways they can be screened?
Colony hybridisation (blotting)
PCR-based screening
immunochemical assays
functional assays (ie an enzyme screened for using antibodies)
Explain how library screening by colony lift is done?
Colonies produced in petri dishes can be stuck to membranes by blotting. These cells are lysed, DNA denatured and fixed onto the membrane.
Then incubated with labelled probes and later wash off any unhybridised probes. The final DNA is hybridised.
What is the principle behind pooling clones of DNA?
Clones are pooled to reduce the number of reactions needed to identify the clones with the target sequence. PCR is used to screen for positive pools with the relevent target sequence.
What is the equation to work out the number of clones needed to get a whole genomic sequence into a library, in order to be able to represent all sequences in the genome?
N= In(1-P) / In(1-f)
N- number of clones
P= probability of finding a particular sequence
f= ratio of the length of the insert in comparison to the entire genome
How many clones are required for a representative genomic library of E.coli?
(gDNA 3.75 x10^6 bp)
assume average length of insert is 20kb and the probability is 0.99.
Ratio= 20,000/3.75x10^6 N= In(1-0.99) / In(1-5.33x10^-3) N= -4.605 / -5.34x10^-3 N= 862 clones required
How many clones are required for a representative genomic library of S. cerevisiae?
(gDNA 1.5x10^7 bp)
assume average length of insert is 20kb and the probability is 0.99.
N= In(1-0.99) / In(1- (20,000/1.5x10^7)) N= 3452 clones required
How many clones are required for a representative genomic library of H. sapiens?
(gDNA 3x10^9 bp)
assume average length of insert is 20kb and the probability is 0.99.
N= In(1-0.99) / In(1- (20,000/3x10^9)) N= 690773 clones required
