Maxam - Gilbert Sequencing

Chemical cleavage method

By Meet Patel

(Maxam-Gilbert Sequencing method)

Introduction

In 1980, two American molecular biologists Allan Maxam and Walter gilbert were developed a DNA sequencing method. This method is based on partial base modification and subsequent cleavage of the DNA backbone. Thus the method becomes famous as the “chemical cleavage method” for DNA sequencing.

Principle

Specific chemicals react on purine and pyrimidine bases and modified them. Because of partial modification DNA backbone cleaved and produces various sizes of DNA fragments which are separated in gel electrophoresis based on their size. The 5’end-labeled with the radioactive element is detected in autoradiography.

Method

Basically this method is divided into 5 steps which are following:

1. End labeling with ³²P (radioactive phosphate)
2. Denaturation
3. Chemical treatment
4. Separation on polyacrylamide gel
5. Autoradiography

Let’s see all the steps in detail,

1. End labeling with ³²P (radioactive phosphate): Firstly, DNA was isolated and purified. After the purification DNA fragment must be labeled with the radioactive element at the 5’-end (importance of 5’-end labeling is discus in autoradiography.). Here we use ³²P (an isotope of phosphate) as the radioactive element. The enzyme named alkaline phosphatase catalyzed removal of a phosphate group at 5’-end of DNA and produce 5’-OH group. Now with help of enzyme polynucleotide kinase and radioactive nucleotide (NTP have radioactive phosphate at ϒ position) add radioactive phosphate group at 5’-end.

   

   (Image 1. 5'-end modification of DNA molecule.)

2. Denaturation: denaturation is the process in which two DNA strands are separated and become single-stranded DNA molecules. Basically, DNA molecule is highly stable at normal (RT and 7.0 pH), but in high temperature or due to variation in pH DNA get denature and produce ssDNA molecule. Here we use NaOH as an alkaline solution to increase pH (or HCl as an acid solution to decrease pH.). NaOH disturbed the H-bond between two DNA strands and break the H-bond. Then separate the denature DNA sample. Here GC rich strands are heavier than AT-rich strands. Thus the GC-rich strand moves slowly and the AT-rich strand moves faster. After separation decide which strand you want to sequence and cut the gel piece containing an interesting strand. Then elute out DNA fragments and purified them.

   

   (Image 2. NaOH disturbed H-bond present between two DNA strands and break down the H-bond.)

3. Chemical treatment process: In this process, take four vials and labeled them as “G”, “A+G”, “T+C”, and “C” respectively. Now add DNA samples in equal amounts in each. Then add specific chemicals concerning the flowing table.

   

   (Image 3. vials containing DNA molecule, buffer, and chemical reagents.)

   BASE	CHEMICAL REAGENT	DESCRIPTION
   G	Dimethyl Sulphate (DMS) + Piperidine	DMS methylated Guanine at 7 positions of N-base and piperidine cleave the N-base selectively.
   A+G	DMS + HCl	DMS methylated both the purine bases and cleave just before the base.
   T+C	Hydrazine + Piperidine	Hydrazine protonated N-base and form nick just before base.
   T	Hydrazine + 0.1M NaCl	Protonated cytosine base and cleave it just before base.

   (After this process wash reagent and purified DNA fragments.)
4. Separation on polyacrylamide gel: gel has 4 wells and wells are labeled as “G”, “A+G”, “T+C” and “C”. Then add DNA sample from vials accordingly and allow it to separate out DNA molecule on the bases of size i.e. smallest DNA molecule move faster and larger DNA molecule move slowly. The smallest DNA molecule is at bottom of the gel matrix and the larger DNA molecule is at the top (origin) of the gel matrix. Here, the problem with gel visualization, at the time of chemical cleavage process DNA fragmented in various size with 5’-P* (radioactive phosphate) group as well as without 5’P* group. When we separate chemically cleaved DNA fragments, they produce smear on gel matrix which cannot be distinguished directly. It needs something else for detection. For solving this problem we performed autoradiography.
5. Autoradiography: In this step, we can detect only DNA fragments that have 5’-P* group, and other DNA fragments which do not have 5’-P*, are become invisible on autoradiograph. Autoradiography is an imaging process in which gel is placed between the X-ray film c. Then expose this caste in the UV radiation, due to radiation radioactive phosphate gets energy from the UV source and reaches a higher level of energy, and emits light having a particular wavelength. This light, which is emitted by radioactive phosphate, oxidizes Ag (Silver) coated on the X-ray film and produces a negative image. This image documented and determine the sequence in a particular manner.

   

   (Image 4. autoradiograph. radioactive elements consume UV light and become excited and emit light having particular wavelength are oxidize Ag coated on X-ray film.)

How can detect sequence in the maxam-gilbert method?

For understanding assumes, we have a DNA fragment with sequence 5’-CCGGCGCAGAAGCGGC-ATC-3’. Now in the image, there are four lanes named “G”, “A+T”, “T+C” and “C”. After gel electrophoresis we can see a bend pattern on the autoradiograph, in which last (at the bottom) two bends are present in the lane of “T+C” as well as “C” i.e. the first nucleotide is C (cytosine) and similarly at second last two bends are present same two lanes i.e. the second nucleotide is also C. at third last two bends are present in the lane of “G” as well as “A+G” i.e. the third nucleotide is G (guanine). Again the fourth nucleotide is G and so on. Now see at number 8, single bend present in “A+G” lane but not in “G” lane i.e. the 8^th nucleotide is A (adenine). Now see at number 18, single bends are present in the “T+C” lane but not in the “C” lane i.e. 18^th nucleotide is T (thymine). Similarly get all the nucleotide sequences in this way and we get hole nucleotide sequences which are the same as the sequence which is we assume.

Application

1. Use to get the sequence of particular gene and mapping.
2. To identify the mutation in coding as well as the non-coding region.
3. Identify spices in the world of bacteriology and virology.
4. Use for study evolutionary relationship between two or more spices.
5. Use in various fields like recombination DNA technology, epigenetic and molecular biology.