Biotechnology section.docx - Biotechnology The genome is all the genetic material contained within an organism or cell Gel electrophoresis is used to

Biotechnology section.docx - Biotechnology The genome is...

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Biotechnology The genome is all the genetic material contained within an organism or cell. Gel electrophoresis is used to separate DNA samples based on their size. To carry out gel electrophoresis agarose gel is required, along with a buffer solution, an electric current, and DNA samples. DNA is negatively charged, so they move from the positive terminal towards the negative terminal of the electric current through the agarose gel. Agarose gel is porous. Smaller DNA samples move through the gel at a faster rate than large samples. A comparison ladder with a known number of base pairs may sometimes be used for comparison purposes. At the end of the trial the DNA fragments are spread out according to their size (i.e. number of base pairs.) A sample of interest then may be isolated so it can be copied using PCR or recombinant DNA. PCR stands for polymerase chain reaction . It amplifies a small DNA sample to make many copies. PCR requires the ‘ingredients’ Taq polymerase, free nucleotides, a section of DNA containing the gene of interest, 2 different DNA primers (to bracket the gene of interest), a buffer solution, and a thermocycler machine. The first step is denaturing . The temperature is set to 92°C. The hydrogen bonds of the DNA section are broken, leaving two single strands. The second step is annealing . The temperature is set to 65°C. This allows the two primers to anneal to the ends of the DNA strands according to complimentary base pairing rules. The third step is extension . The Taq polymerase extends the DNA strand by adding free nucleotides, according to complimentary base pairing. These steps can be repeated to produce a large amount of copies of a single section of DNA within a relatively short time. Making many copies of a DNA sample is also called amplifying . DNA sequencing is highly similar to PCR. DNA sequencing uses forms of nucleotides called dideoxynucleotides ( ddNTPs .) Once a dideoxynucleotide has been added to a chain, no more nucleotides can be added. They also contain a fluorescent marker. A number of different lengths of DNA are created as a result of the addition of these nucleotides. The fragments are then run through gel electrophoresis. A laser reads the fluorescent marker and sequences the DNA. o Originally, four different ddNTPs were used in four different solutions. o The fragments were then run through gel electrophoresis and the bases had to be manually sequenced. o This technique is known as Sanger sequencing and it is still used for smaller sequencing projects. DNA sequencing has applications in comparative genomics where the entire genomes of different species are sequenced and compared.
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DNA transformation uses bacterial plasmids in order to create many copies of a gene of interest.
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  • Fall '18
  • maya

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