Maxam–Gilbert sequencing

Maxam-Gilbert sequencingMaxam-Gilbert
Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1977–1980.wikipedia
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Walter Gilbert

GilbertGilbert, WalterWalter (Wally) Gilbert
Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1977–1980.
Together with Allan Maxam, Gilbert developed a new DNA sequencing method, Maxam–Gilbert sequencing, using chemical methods developed by Andrei Mirzabekov.

Allan Maxam

Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1977–1980.
Walter Gilbert and Allan Maxam developed a DNA sequencing method - now called Maxam-Gilbert sequencing - which combined chemicals that cut DNA only at specific bases with radioactive labeling and polyacrylamide gel electrophoresis to determine the sequence of long DNA segments.

DNA sequencing

DNA sequencesequencesequencing
Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1977–1980.

Nucleobase

basesnucleobasesbase
This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.

Bond cleavage

cleavagecleavedcleave
This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.

Nucleotide

nucleotidesntdinucleotide
This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.

Sanger sequencing

chain termination methodSanger methodSanger
Maxam–Gilbert sequencing was the first widely adopted method for DNA sequencing, and, along with the Sanger dideoxy method, represents the first generation of DNA sequencing methods.

Frederick Sanger

Fred SangerSangerFrederic Sanger
Although Maxam and Gilbert published their chemical sequencing method two years after Frederick Sanger and Alan Coulson published their work on plus-minus sequencing, Maxam–Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA.

Molecular cloning

cloneclonedrecombinant DNA technology
Although Maxam and Gilbert published their chemical sequencing method two years after Frederick Sanger and Alan Coulson published their work on plus-minus sequencing, Maxam–Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA.

Radioactive tracer

radiotracerradiolabeledradiolabel
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma- 32 P ATP) and purification of the DNA.

Directionality (molecular biology)

533' end
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma- 32 P ATP) and purification of the DNA.

Kinase

kinaseskinase domainprotein kinase C
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma- 32 P ATP) and purification of the DNA.

Phosphorus-32

32 PP3232P
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma- 32 P ATP) and purification of the DNA.

Adenosine triphosphate

ATPadenosine triphosphate (ATP)adenosine 5'-triphosphate
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma- 32 P ATP) and purification of the DNA.

Purine

purinespu'''R'''ineTraube purine synthesis
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Formic acid

formicformateHCO 2 H
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Guanine

GG'''uanineGG
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Adenine

AA'''denineadenine nucleotides
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Dimethyl sulfate

dimethylsulfate(CH 3 O) 2 SO 2 (CH 3 ) 2 SO 4
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Pyrimidine

pyrimidinespyrimidine nucleotidesp'''Y'''rimidine
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Hydrazine

hydrazine hydratehydraziniumN 2 H 4
For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine.

Sodium chloride

NaClsaltroad salt
The addition of salt (sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for the C-only reaction.

Piperidine

piperidinespiperidinylNC5H10
The modified DNAs may then be cleaved by hot piperidine; (CH 2 ) 5 NH at the position of the modified base.

Gel electrophoresis

gelelectrophoresiselectrophoresis gel
The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation.

Autoradiograph

autoradiographyautoradiographicautoradiographs
To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each showing the location of identical radiolabeled DNA molecules.