Lecture 25 Flashcards
How are BAC vectors used to construct a genomic library?
DNA is cut with blunt cutting
and isolated into large fragments via gel
Random shearing is used to ensure random even coverage of the genome
Vector is cut with use of a phosphatase to stop recircularization
DNA is stored as cells in ordered arrays
What is a cDNA library?
Set of clones which contain all the mRNAs expressed in a certain cell type of an organism
However early cDNA clones often have incomplete 5’ ends due to premature termination of reverse transcriptase
or loss of bases at 5’ cap due to polishing
How is a cDNA library constructed?
A population of mRNA is isolated through an oligo DT column
An oligo-DT primer is annealed allowing Reverse transcriptase to produce a DNA copy
RNAseH is then used to attack the RNA but leave the DNA intact
DNA pol. 1 is then used to make double stranded DNA
This is then cloned into vectors
What are the differences between a genomic and cDNA library?
The source of genomic libraries is chromosomal DNA while cDNA uses mRNA
Genomic libraries contain all regions including regulatory regions like promoters and introns not seen in cDNA libraries
cDNA uses only one gene per clone while genomic libraries use large chunks
What is the catch 22 around PCR?
While it has largely replaced the construction of libraries and screening, the technique requires something about the sequence to be known in order to design the correct primers
How large is a mammalian genome?
3*10^9 br or 21,000 genes
How large is the average BAC insert?
100 kb
How many times coverage of the organisms genome has to be made to ensure it is representative?
4.6 times coverage
How many mRNAs are in a typical mammalian cell?
2*10^5 mRNAs
How large does a cDNA library need to be to be representative?
1 million clones
What is the variance of mRNA copies per cell?
some genes have only 1 mRNA per 10 cells while some have 100 000 copies in just one cell
What are the three methods of finding a gene from a library?
Colony or plaque hybridisation through use of a labelled probe
Antibody detection of an expressed protein
Complementation of E.Coli or yeast mutants
How can colony/plaque hybridization with a probe be used to find a gene?
A primer must be identified through:
Using the gene of another species and identifying under low stringency
Using the known protein we can make a probe of the corresponding sequence
How can an antibody detection be used to find a gene?
If the purified protein is known then an expression library can be constructed and antibodies are used to identify if the protein is being expressed
