Ajay Ramgolam (USI:1038880)
WEEK 7 Discussion questions
1. Why are restriction enzymes useful to the molecular biologist?
Restriction enzymes have proved to be invaluable for the physical mapping of DNA.
They offer unparalleled opportunities for diagnosing DNA sequence content and are used
in fields as disparate as criminal forensics and basic research. In fact, without restriction
enzymes, the biotechnology industry would certainly not have flourished as it has. The
first experiments demonstrating the utility of restriction enzymes were carried out by
Danna and Nathans and reported in 1971. This pioneering study set the stage for the
modern practice of molecular biology in which restriction enzymes are ubiquitous tools,
although they are often taken for granted.
2. What is the basis for separating DNA molecules by electrophoresis?
All DNA molecules have the same amount of charge per mass. Because of this, gel
electrophoresis of DNA fragments separates them based on size only. Using electrophoresis, we
can see how many different DNA fragments are present in a sample and how large they are
relative to one another.
3. Why is a primer needed at each end of the DNA segment being amplified by PCR?
Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add
DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase
enzyme attach and start making the new complementary strand of DNA from the loose DNA
bases.
4. What are the roles of a cloning vector, restriction enzymes, and
DNA ligase in molecular cloning?
Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a
few target sequences and cuts DNA at or near those sequences.
Many restriction enzymes make staggered cuts, producing ends with single-stranded
DNA overhangs. However, some produce blunt ends.
DNA ligase is a DNA-joining enzyme. If two pieces of DNA have matching ends,
ligase can link them to form a single, unbroken molecule of DNA.
In DNA cloning, restriction enzymes and DNA ligase are used to insert genes and
other pieces of DNA into plasmids.
5. How may cloned genes be identified? When would it be beneficial to use a
eukaryotic host for molecular cloning?
DNA cloning is the process of making multiple, identical copies of a particular piece of
DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest
(perhaps a gene for a medically important human protein) is first inserted into a circular
piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste”
DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of
fragments from multiple sources.
6. What major advantage does cloning mammalian genes from mRNA or using
synthetic genes have over PCR amplification and cloning of the native gene?
Both PCR and molecular cloning can be used to direct the replication of DNA sequences.
There are several fundamental differences between these two methods.
Molecular cloning replicates DNA within in a living cell, while PCR replicates DNA in
an in vitro solution, free of living cells.
Molecular cloning involves cutting and pasting the sequences, while PCR amplifies DNA
by copying an existing sequence.
DNA cloned by molecular cloning is usually faithfully copied and fully functional,
whereas PCR introduces errors in sequence, resulting in mutations.
7. How is a fusion protein made?
Natural occurrence. Naturally occurring fusion genes are most commonly created when a
chromosomal translocation replaces the terminal exons of one gene with intact exons
from a second gene. This creates a single gene that can be transcribed, spliced, and
translated to produce a functional fusion protein.
8. What do the acronyms BAC and YAC stand for?
YAC stands for Yeast Artificial Chromosome and BAC stands for Bacterial artificial
chromosome.