For unlimited access to Study Guides, a Grade+ subscription is required.
Molecular Genetics Methodologies II
Some historical perspective into E.coli and bacteriophages:
Dr. Paul Berg and others were the first to successfully introduce exogenous DNA fragments into
bacteria to produce a recombinant gene product Uprising of Recombinant DNA technology
Bacterial restriction enzymes cut DNA at
Polylinkers are engineered with many restriction
enzyme sites to facilitate insertion of DNA.
EcoRI cuts DNA at a specific palindromic sequence, GAATC. Note: Vector is a specialized plasmid, which carries out
specific functions upon removal of nonfunctional regions.
However, if the Polylinker undergoes digestion by
2 different enzymes, direction cloning results, thus
it only inserts in one orientation.
DNA libraries (permanent collection of genes):
1. Genomic library contain copies of DNA present in the genomic/chromosomal context
(intergenic regions, introns, exons, repetitive sequence)
2. cDNA library represent mRNA (tissue specificity, abundance)
After RT uses oligo-T primer (polydT) to initiate the ssDNA
synthesis that is complementary to the mRNA template, RNA
is then removed and a polydG adapter(GGG) is annealed to
the 3’ end. A polydC primer(CCC) is used to initiate synthesis
of the 2nd DNA strand.
E.coli DNA polymerase I
progresses to extend any
remaining hybrid regions.
(DNA synthesis stops at
the 3’ end)
Note: Alkaline may serve
as RNase H to break
hydrogen bonds and
degrade RNA. Then
PolydG and PolydC are
Almost no one uses cDNA libraries now...because...
Studying Genes of interest: RT-PCR
Since all mRNAs present at a given cell will have a
poly(A) tail, then poly T primers can be used to
prime a cDNA using RT-PCR.
The resulting cDNA molecules in a reaction
solution after several cycles should be a
reasonable prediction of various starting levels of
each species. The population can be screened
using degenerate primers.
Bacterial Expression Vectors: over expression of recombinant protein (ex. Insulin)
The lacZ promoter(under control of IPTG) provides
an effective means of inducing gene expression.
For example: it can drive exogenous T7 RNA
polymerase, which expresses recombinant gene
under the control of T7 promoter.
Endogenous: native to the organism.
Exogenous: introduced from another organism.