Sequences of DNA allows us to provide a full analysis of DNA, because it gives us basic information on all: sequence of nucleotides. With this knowledge, for example, we can organize and track gensequenties to make comparisons between homologous genes to identify all types of mutations. Scientists admit that he was potentially a very powerful tool, and thus had no competition to create a method to sequence DNA. Then in 1974, two methods were developed separately by an American team and an English team to do exactly that. Americans, led by Maxam and Gilbert, using a chemical cleavage protocol, while in English, led by Sanger, design a procedure similar to the natural process of DNA replication. Although the two teams shared the Nobel Prize in 1980, Sanger method became standard because of its practical (fast, 1992).
Sanger's method, also referred to as sequencing or dideoxy chain termination, is based on the use of dideoxynucleotides (ddNTP's) than normal nucleotides (NTP) in the DNA results.Dideoxynucleotides is essentially the same as nucleotides except it contains a group of hydrogen to carbon 3 'instead of a hydroxyl group (OH). This nucleotides change, when integrated in a row, so that further addition of nucleotides. (Speed, 1992). This has happened because of a link between phosphodiester can create and dideoxynucleotide next next nucleotide, and thus the DNA chain is terminated.
Method
Before DNA sequences may be, it must be denatured into single strands using heat. Together, a primer is annealed to a template strands. This primer is specifically designed so that the end 3 'of its DNA sequences along the interest is determined. Or is a primer or nucleotides be a radioactive or fluorescently labeled so that the final product on a gel (Russell, 2002) will be revealed. After the primer is attached to DNA, the solution is divided into four tubes with the name "G", "n", "T" and "C". Then reagents were added to these samples as follows:
"G" tube: All four dNTP's, ddGTP and DNA polymerase
"A" tube: All four dNTP's, ddATP and DNA polymerase
"" T tube: All four dNTP's, ddTTP and DNA polymerase
"C" tube: All four of dNTP, ddCTP and DNA polymerase
As seen above, all tubes containing a different ddNTP is present, each about a hundreth of a normal concentration of precursors (Russell, 2002). As synthesized DNA, nucleotides are added to the growing chain of DNA polymerase. However, sometimes in the dideoxynucleotide is involved in the chain instead of a normal nucleotide, which leads to a chain-terminating event.For example, if we consider only "G pipe", perhaps we find a mixture of the following products:
Key to this method is that all responses to the same nucleotide starts and ends with a special basis. So, in a country where same-synthesized DNA chain break, the new chain will be completed in all nucleotide positions where there is potential to increase as a result of incorporation of dideoxynucleotides (Russell, 2002). In this way, bands of all different lengths are produced. After the reaction is complete, the DNA is denatured again in preparation for electrophoresis. The content of each of four tubes in each job to run for a gel to separate different size hoops polyacrylmide away. Once content is done through the gel, the gel is then exposed or UV light or X-Ray, depending on the method used for labeling DNA.
Figure 2: This is a gel tube polyacrylmide reactions in "G" (the same sequences shown in Figure 1). DNA fragments on the travel distance shorter than small fragments due to most of their heavy blue indicates molecular weight.The primer, black part shows the newly synthesized chain and red indicates a ddGTP, the chain ( Metzenberg) terminated.
As shown in Figure 2, small fragments are produced when closer ddNTP primer will be added, because the chain is smaller and therefore migrated faster on the gel. Once all the responses from the four tubes in a gel, the DNA sequences present in the direction 5 'to 3 "can be combined model of reading at the end of gel rings to be determined. It is important to remember that although this series is complementary to the template strand from the beginning.
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