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DNA Sequencing: Maxam Gilbert MethodBY: Farah Mahboob | Category: Biotech-Research | Submitted: 2010-07-21 04:08:56
Maxam Gilbert Method:
Through this technique the two scientists reported the sequence of 24 base pairs nucleotide sequence of a lac operator. Although their paper was published two years after the famous Sanger method, still their technique became more popular. The process uses purified DNA directly, chemically modifies the DNA and subsequently cleaves it at specific base sites. The process is listed below in six steps;
Step 1: Purifying the Sequence
1. Enzyme, restriction endonuclease is used and DNA is cut at a specific sequence. For example, if the restriction endonuclease is 'Hind lll', it is responsible for cleaving the sequence AAGCTT.
Step 2: Addition of radioactive phosphate
2. Since DNA has sugar phosphate back bone, phosphate present at the 3' end of the cleaved DNA segment will be removed and replaced by radioactive phosphate (32p).
3. Phosphatase is the enzyme for phosphate cleavage while Kinase is the enzyme used for radioactive phosphate addition.
Step 3: Seperating the sub fragments
4. The radioactive labelled DNA fragment is again treated with another restriction endonuclease. This endonuclease further cuts the DNA fragment.
5. DNA fragments are ran through Gel electrophoresis to separate the two, labeled and unlabeled-end sub fragments from each other resulting in sub fragments having one labeled and an unlabeled end.
6. The DNA sub fragment whose sequence is to be determined is purified from the gel and separated from its other end-labeled sub fragment.
Step 4: Identifying the Bases
7. Four base specific chemical samples are produced. For example, chemical sample for Guanine will cause the bond holding the base Guanine in position of the DNA to break. Similarly other chemicals break the bonds holding bases Cytosine and another breaking both Adenine with some cleavage or weakening of Adenine, the fourth one breaks the bonds holding the Thymine with some cleavage or weakening of Cytosine bases. Thus, the four reaction samples are;
1. G reaction (dimethyl sulfate (DMS) methylates Guanine).
2. C reaction.
3. A reaction with some G cleavage (DMS also methylates Adenine but does not result is strand cleavege).
4. T reaction with some C cleavage.
For G reaction Piperidine is used. This causes loss of the methylated base and breakage of DNA backbone at the lost base site. The sites are called apurinic site. For Adenine and Guanine glycoside bonds can also be weaken with acid and later on piperidine used that causes depurination and strand breakage.
For Thymine and Cytosine, hydrazine is used which open up their rings. Later on piperidine is used to create apyrumidinic sites by cleaving the bases and breaking the back bone.
On purines Adenine and Guanine cleavage apurinic sites are created where as, for pyrimidines, cytosine and thymine cleavage apyrumidinic sites are created.
8. The end-labeled DNA fragment are further divided and placed in these four separate chemical solutions.
9. As explained earlier, each reaction solution only treats a particular base therefore, for example in G reaction solution, each DNA molecule will only have its Guanine bond broken and the base removed.
10. In this way every Guanine base in the DNA molecule will be removed either if its 100 bases away or at the end of the molecule.
Step 5: Cleaving the DNA
11. When the bases are removed for each particular base removing reaction the DNA strands are subjected to another reagent. This reagent breaks the DNA at the very particular points from where the bases have been removed.
12. This results in DNA strands of different lengths.
Step 6: Reading the Sequence
13. Electrophoresis is performed again on the four reaction samples.
14. Each reaction is ran on its own lane and arranged according to its length.
15. Autoradiography: a technique that reads radioactive molecules on an x-ray film, is used to detect the separated DNA fragments.
16. On reading the X-ray films, the bands of DNA fragments are revealed according to their length in each separated lane of the four reaction mixtures.
A Reaction with some G cleavage
9 - - - - - -
3 - - - - - -
T Reaction with some C Cleavage
11 - - - - - -
10 - - - - - -
5 - - - - - -
Base pairs Identified against each reaction mixture
"=====" represent a dark band, "- - - - - -" represents a light band as seen on the X-ray film .
Thus the Sequence is read from down to up, as TTGACTTAGCC. A dark band in each reaction depicts the base present. A band found in the A reaction with some cleavage at G is read as 'A', that is if there is no corresponding band of the same length in the G lane. Same goes for the T reaction with some G cleavage.
Although this process was more popular as compared to Sanger method, Maxam and Gilbert method did not remain on scientists favourite DNA sequencing techniques list for long, due to its technical complexity and extensive use of hazardous material.
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