第一代测序 Sanger法DNA测序流程图 （英文）
(2011-10-16 12:22:47)
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DNA quencing enables us to perform a thorough analysis of DNA becau it provides us with the most basic information of all: the quence of nucleotides. With this knowledge, for example, we can locate regulatory and gene quences, make comparisons between homologous genes across species and identify mutations. Scientists recognized that this could potentially be a very powerful tool, and so there was competition to create a method that would quence DNA. Then in 1974, two methods were independently developed by an American team and an English team to do exactly this. The Americans, lead by Maxam and Gilbert, ud a “chemical cleavage protocol”, while the English, lead by Sanger, designed a procedure similar to the natural process of DNA replication. Even though both teams shared the 1980 Nobel Prize, Sanger’s method became the standard becau of its practicality (Speed, 1992).
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Sanger’s method, which is also referred to as dideoxy quencing or chain termination, is b
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ad on the u of dideoxynucleotides (ddNTP’s) in addition to the normal nucleotides (NTP’s) found in DNA. Dideoxynucleotides are esntially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). The modified nucleotides, when integrated into a quence, prevent the addition of further nucleotides. (Speed, 1992).This occurs becau a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.
The Method
Before the DNA can be quenced, it has to be denatured into single strands using heat. Next a primer is annealed to one of the template strands. This primer is specifically constructed so that its 3' end is located next to the DNA quence of interest. Either this primer or one of the nucleotides should be radioactively or fluorescently labeled so that the final product can be detected on a gel (Rusll, 2002). Once the primer is attached to the DNA, the solution is divided into four tubes labeled "G", "A", "T" and "C". Then reagents are added to the samples as follows:
"G" tube: all four dNTP's, anyway什么意思ddGTP and DNA polymerabass是什么意思
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"A" tube: all four dNTP's, ddATP and DNA polymera
"T" tube:我想见你的英文 all four dNTP's, ddTTP and DNA polymera
"C" tube: all four dNTP's, 深圳翻译ddCTP 怀恩豪斯and DNA polymera
As shown above, all of the tubes contain a different ddNTP prent, and each at about one-hundreth the concentration of the the normal precursors (Rusll, 2002). As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA polymera. However, on occasion a dideoxynucleotide is incorporated into the chain in place of a normal nucleotide, which results in a chain-terminating event. For example if we looked at only the "G" tube, we might find a mixture of the following products:
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Figure 1: An example of the potential fragments that could be produced in the "G" tube. The fragments are all different lengths due to the random integration of the ddGTP's (Metzenberg).
The key to this method, is that all the reactions start from the same nucleotide and end with a specific ba. Thus in a solution where the same chain of DNA is being synthesized over and over again, the new chain will terminate at all positions where the n
ucleotide has the potential to be added becau of the integration of the dideoxynucleotides (Rusll, 2002). In this way, bands of all different lengths are produced. Once the reactions are completed, the DNA is once again denatured in preparation for electrophoresis. The contents of each of the four tubes are run in parate lanes on a polyacrylmide gel in order to parate the different sized bands from one another. After the contents have been run across the gel, the gel is then expod to either UV light or X-Ray, depending on the method ud for labeling the DNA.
Figure 2: This is a polyacrylmide gel of the reactions in the "G" tube (the same quence
s en in figure 1). The longer fragments of DNA traveled shorter distances than the smaller fragments becau of their heavier molecular weight.The blue ction indicates the primer, the black ction indicates the newly synthesized strand and the red denotes a ddGTP, which terminated the chain (Metzenberg).
As shown in Figure 2, smaller fragments are produced when the ddNTP is added clor to the primer becau the chains are smaller and therefore migrate faster across the gel. If all of the reactions from the four tubes are combined on one gel, the actual DNA quence in the 5' to 3' direction can be determined by reading the banding pattern from the bottom of the gel up. It is important to remember though that this quence is complementary to the template strand from the beginning.
