Dna Sequencing Lab Report

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The Sanger Method of DNA Sequencing Introduction: The sanger method which is also known as the chain terminating method or dideoxy sequencing is a method of DNA sequencing requiring the addition of chain-terminating dideoxynucleotides (ddNTP's) by the enzyme DNA polymerase during DNA replication. This method was an idea by the scientist Frederick Sanger in 1977 and was the most common method of DNA sequencing for 25 years. Fred Sanger and his two co-workers, Aaron Klug and Alan Coulson, came up with a way of gene sequencing which was the first of its kind. It was relatively rapid; they could sequence a few thousand base quite once you had your gene. What they could not do was sequence a hundred million bases of the worm genome at that time.…show more content…
This is done by heating the double-stranded DNA to denature it. This then splits the DNA into two single-stranded pieces of DNA. A primer is then annealed to the end of one of the template strand of DNA. This primer is made so that its 3' end is located exactly next to the DNA sequence we are interested in. This DNA is then placed in four different test-tubes. DNA polymerase is added to each tube along with dNTP's. Then chain-terminating dideoxynucleotides (ddNTP's) are added to each tube, a different one in each. Dideoxy ATP is added to the first, dideoxy TTP to the second, dideoxy CTP to the third and dideoxy GTP to the fourth tube. Now the samples are ready for separation using polyacrymide gel electrophoresis. A sample of each is pipetted into a different lane on the electrophoresis machine. Electric current is added and the bases are separated based on size, the smallest/lightest pieces moving the furthest down the machine and the largest/heaviest pieces staying close to the beginning of each lane. Either UV light or X-ray is shone on the gel, depending on which method is being used to label the DNA sequence. The sequence is read from the bottom of the gel to top in terms of nucleotide bases e.g. ATCTGAT. With the many huge improvements in technology that have been achieved since 1974, it isn’t a shock that the Sanger method has become out of date. Despite this, nearly all the new technology that has…show more content…
This method was an idea by the scientist Frederick Sanger in 1977 and was the most common method of DNA sequencing for 25 years. Fred Sanger and his two co-workers, Aaron Klug and Alan Coulson, came up with a way of gene sequencing which was the first of its kind. It was relatively rapid; they could sequence a few thousand base quite once you had your gene. What they could not do was sequence a hundred million bases of the worm genome at that time. The chain was just too long. The problem of isolating a specific section of genes still existed. What it means to isolate a gene is to somehow cut out the relevant piece of the whole genome, place it into a bacterium, clone it, and then you could have indefinite amounts of this particular piece as the bacteria replicates. Dideoxynucleotides are approximately the same as nucleotides apart from one difference, that they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH) on the same carbon. These modified nucleotides, when joined into a DNA sequence, prevent the addition of any more nucleotides. This happens as a phosphodiester bond cannot be constructed between a ddNTP and the next nucleotide. The chain then ends and the sequence of bases can be uncovered by a few
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