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Biophysics Assignment 2 PDF

The Maxam-Gilbert sequencing method involves: 1) Labeling DNA fragments with radioactive phosphates 2) Treating the labeled fragments separately with base-specific chemicals that cleave the DNA at specific bases 3) Separating the cleaved fragments by size via gel electrophoresis and detecting the fragments using autoradiography.

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179 views

Biophysics Assignment 2 PDF

The Maxam-Gilbert sequencing method involves: 1) Labeling DNA fragments with radioactive phosphates 2) Treating the labeled fragments separately with base-specific chemicals that cleave the DNA at specific bases 3) Separating the cleaved fragments by size via gel electrophoresis and detecting the fragments using autoradiography.

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Kwasi Bempong
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© © All Rights Reserved
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① In the Maxam-Gilbert procedure, the DNA fragment is labelled with 32P at its 5' end.

Then chemicals are used that break the DNA preferentially at each of the four
nucleotide bases under conditions in which only one break per chain is made. Thus
four separate test tubes are prepared, one for each base (A, T, G, C). After gel
electrophoresis and autoradiography only the fragments possessing 5'-terminal 32P-
phosphate group show up on the gel. This chemical method is only seldom used
because it is time-consuming and requires handling of toxic chemicals.
Simply put, Maxam Gilbert sequencing method is performed via several steps as

i
follows.

Purification of the DNA sequence using restriction endonucleases


Labeling of the ends of the DNA fragments by adding radioactive phosphates
Purification of the labeled fragments from non-labeled fragments by gel
electrophoresis
Separation of the end-labeled DNA into four tubes and treating with base specific
chemicals separately
Electrophoresis of the contents of each tube on separate lines on a gel and fragment
separation according to their length.
Detection of the fragments by autoradiograph.
.

slit Purines will react with dimethyl sulfate and pyrimidines will react with hydrazine in
such a way as to break the glycoside bond between the ribose sugar and the base
displacing the base
Piperidine will then catalyze phosphodiester bond cleavage where the base has been
displaced

÷
Dimethyl sulfate and piperidine alone will selectively cleave guanine nucleotides but
dimethyl sulfate and piperidine in formic acid will cleave both guanine and adenine
nucleotides. Similarly, hydrazine and piperidine will cleave both thymine and cytosine
nucleotides whereas hydrazine and piperidine in 1.5M NaCl will only cleave cytosine
nucleotides

Gel electrophoresis is a technique commonly used in laboratories to separate charged


molecules like DNA, RNA and proteins according to their size.
Charged molecules move through a gel when an electric current is passed across it.
An electric current is applied across the gel so that one end of the gel has a positive
charge and the other end has a negative charge.
The movement of charged molecules is called migration. Molecules migrate towards
the opposite charge. A molecule with a negative charge will therefore be pulled
towards the positive end (opposites attract!).
The gel consists of a permeable matrix, a bit like a sieve, through which molecules
can travel when an electric current is passed across it.
Smaller molecules migrate through the gel more quickly and therefore travel further
than larger fragments that migrate more slowly and therefore will travel a shorter
distance. As a result the molecules are separated by size. Electrophoresis enables you
to distinguish DNA fragments of different lengths. DNA is negatively charged,
therefore, when an electric current is applied to the gel, DNA will migrate towards the
positively charged electrode. Shorter strands of DNA move more quickly through the
gel than longer strands resulting in the fragments being arranged in order of size.

3) The Maxam-Gilbert method used a large amount of a radioisotope for labeling DNA
molecules, and the reagent used for chemical cleavage was highly poisonous and
unstable.
④ Differences

similarities

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