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The Use of a Bacterial Plasmid to Clone and Sequence a Human Gene

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#1. a) The Use of a Bacterial Plasmid to Clone and Sequence a Human Gene

The process begins with restriction endonucleases scanning and binding to double-stranded DNA at specific base-pair sequences, the recognition sites, in a predictable manner. The restriction sites are usually 4 to 8 base pairs long and are characterized by the palindromic sequences, with both strands having the same sequence when read in opposite direction. After the restriction endonuclease binds, it starts to disrupt, using hydrolysis, the phosphodiester bonds between neighbor nucleotides, causing the H-bonds between base pairs in the cutting region to be broken. This cuts the original double-stranded DNA strand, producing two DNA fragments, which may differ for different restriction endonucleases, depending on where the phosphodiester bond is broken when cut by the endonuclease. This process can produce either blunt ends (where ends of the DNA fragment are fully paired with no overhangs), or sticky ends (where both DNA fragments have nucleotides lacking complementary bases and overhangs are produced). However, sticky ends are more useful for genetic engineering. The next step, gel electrophoresis, separates the gene that has been excised, from the unwanted fragments taking advantage of chemical and physical properties of DNA. The DNA fragments travel through gel as a result of charge passed through it causing the longer fragments to separate from shorter ones, which helps in identifying gene and makes it easier to cut it out from the gel. The DNA fragment with the desired gene is, therefore, excised and purified. The same restriction endonuclease, that is used to cut the original DNA strand, then splices this gene into a plasmid (small, circular DNA molecules found in bacteria). Because the plasmid and the foreign gene are cut by the same restriction endonuclease, the sticky ends formed, are complementary and anneal to each other forming H-bonds. The DNA ligase reforms the phosphodiester bonds, after which, the recombinant plasmid with the foreign DNA, is introduced into the bacterial cell, in the process of transformation, and replicates to form clones (exact copies of itself). Overall, this technique of altering sequence of DNA molecules became very useful for many reasons, one of which is production of hormones. Hormones such as insulin and somatropin, were produced by inserting a gene into a plasmid, and became essential hormones in medical practice. In this process, the needed human genes were incorporated into plasmids and activated or inactivated when needed, using specific inducers for promoter regions.

Polymerase Chain Reaction

Direct, unlike the use of bacterial plasmid, method of biotechnology to make copies of the desired sequence of DNA. At first, the DNA strands are separated using heat, which causes the H bonds to break between strands. The two strands separate and are used as templates to build complementary strands using the Taf. polymerase (similar to DNA polymerase III, but can withstand high temperatures) and two DNA primers (forward and reverse DNA primers, synthesizing DNA in opposite directions), in a process similar to DNA replication. After the complementary strands are build, the cycle repeats with new double-stranded copies of the targeted DNA build. In this method of biotechnology, however, the targeted areas on DNA is not isolated in first steps. In the first cycle, for example, only variable-length DNA strands are isolated, which start with target region DNA and extend beyond it. In the second cycle, the constant-length DNA strands are produced, which start and terminate at the target region. After the third cycle, the number of copies of the targeted strand only increase exponentially. This cycle technique is useful in forensic criminal investigations, medical diagnostics, genetic research, and many other fields, because it requires only a small part of DNA, and takes relatively little time to accomplish.

#2 a) Gel Electrophoresis is the technique used to separate a gene that has been excised, from the unwanted fragments. It takes advantage of varying length of different DNA fragments and their negative charge. In this process, DNA fragments travel through the gel at different speeds, depending on their size. Shorter fragments travel faster, while longer fragments - slower. Electrical current is used to place negative charge at one end of the gel, where the DNA fragments are placed, while positive charge is placed at the opposite end of the gel. The electrolyte solution causes a current to pass through the gel and negatively charged DNA to migrate toward positively charged electrode.



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