You want to make a recombinant DNA in which aPCR product amplified from the human genome is inserted into a plasmid vector. The polylinker of thisvector includes recognition sites for the enzymesEcoRI (5′ G^AATTC 3′) and BamHI (5′ G^GATCC3′). (The ^ symbolizes the cut site in the DNA.) PCRprimers that could amplify the fragment of humanDNA are: 5′ GCTACTTCGCGTATTCCA 3′ and5′ CCCAAGTCCTAGCCGATA 3′.a. Describe in detail how these primers would need tobe modified to create a fragment of the human genome flanked by EcoRI sticky ends so that thisfragment could be cloned easily into the plasmidvector. You will need to consider the fact that mostrestriction enzymes, including EcoRI, cannot cutDNA if the restriction site is directly at the end ofthe DNA molecule; the restriction enzyme recognition site must be at least six base pairs distant fromthe end.b. Describe a potential feature of the PCR-amplifiedregion of the human genome that could prevent youfrom using the strategy you described in part (a).c. Now describe how the primers must be modifiedto create a human DNA fragment with an EcoRIcompatible single-stranded overhang at one endand a BamHI-compatible overhang at the otherend. (Two possibilities exist; you need todescribe only one. Assume that a BamHI site alsomust be at least six base pairs from the end ofthe DNA.) Why might you want to make sucha fragment?
Bacterial Genomics
The study of the morphological, physiological, and evolutionary aspects of the bacterial genome is referred to as bacterial genomics. This subdisciplinary field aids in understanding how genes are assembled into genomes. Further, bacterial or microbial genomics has helped researchers in understanding the pathogenicity of bacteria and other microbes.
Transformation Experiment in Bacteria
In the discovery of genetic material, the experiment conducted by Frederick Griffith on Streptococcus pneumonia proved to be a stepping stone.
Plasmids and Vectors
The DNA molecule that exists in a circular shape and is smaller in size which is capable of its replication is called Plasmids. In other words, it is called extra-chromosomal plasmid DNA. Vectors are the molecule which is capable of carrying genetic material which can be transferred into another cell and further carry out replication and expression. Plasmids can act as vectors.
You want to make a recombinant DNA in which a
PCR product amplified from the human genome is inserted into a plasmid vector. The polylinker of this
vector includes recognition sites for the enzymes
EcoRI (5′ G^AATTC 3′) and BamHI (5′ G^GATCC
3′). (The ^ symbolizes the cut site in the DNA.) PCR
primers that could amplify the fragment of human
DNA are: 5′ GCTACTTCGCGTATTCCA 3′ and
5′ CCCAAGTCCTAGCCGATA 3′.
a. Describe in detail how these primers would need to
be modified to create a fragment of the human genome flanked by EcoRI sticky ends so that this
fragment could be cloned easily into the plasmid
vector. You will need to consider the fact that most
restriction enzymes, including EcoRI, cannot cut
DNA if the restriction site is directly at the end of
the DNA molecule; the restriction enzyme recognition site must be at least six base pairs distant from
the end.
b. Describe a potential feature of the PCR-amplified
region of the human genome that could prevent you
from using the strategy you described in part (a).
c. Now describe how the primers must be modified
to create a human DNA fragment with an EcoRIcompatible single-stranded overhang at one end
and a BamHI-compatible overhang at the other
end. (Two possibilities exist; you need to
describe only one. Assume that a BamHI site also
must be at least six base pairs from the end of
the DNA.) Why might you want to make such
a fragment?
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