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Chain Termination Method

The Sanger Dideoxy Method, developed by Fred Sanger in 1974, utilizes DNA polymerase and dideoxyribonucleotides to terminate DNA chain elongation during sequencing. The process involves preparing a reaction mixture with template DNA, primers, and nucleotides, which is then divided into four tubes containing different dideoxynucleotides to stop the synthesis at specific points. The resulting DNA fragments are analyzed through electrophoresis and detected via autoradiography.

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20 views1 page

Chain Termination Method

The Sanger Dideoxy Method, developed by Fred Sanger in 1974, utilizes DNA polymerase and dideoxyribonucleotides to terminate DNA chain elongation during sequencing. The process involves preparing a reaction mixture with template DNA, primers, and nucleotides, which is then divided into four tubes containing different dideoxynucleotides to stop the synthesis at specific points. The resulting DNA fragments are analyzed through electrophoresis and detected via autoradiography.

Uploaded by

Upadesh Pattnaik
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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1.

CHAIN TERMINATION METHOD (SANGER DIDEOXY METHOD): This method was developed
by Fred Sanger in 1974, which utilise DNA polymerase to extend DNA chain length. Sanger
used the principle of DNA replication for the development of Dideoxy Sequencing method.
At about the same time as Maxam-Gilbert DNA sequencing was being developed; Fred
Sanger was developing an alternative method. Rather than using chemical cleavage
reactions, Sanger opted for a method involving a third form of the ribose sugars. Ribose has
a hydroxyl group on both the 2’ and the 3’ carbons whereas deoxyribose has only the one
hydroxyl group on the 3’ carbon which is used for DNA synthesis. There is a third form of
ribose in which the hydroxyl group is missing from both the 2’ and the 3’ carbons. This is
dideoxyribose. Sanger knew that whenever a dideoxynucleotide was incorporated into a
polynucleotide, the chain would irreversibly stop, or terminate and (dents) is used as DNA
chain terminators.
2. The enzymatic synthesis method requires 5 things :-1. A short chain primer 2. A ssDNA
templates 3. The 4 terminators (ddATP, ddGTP, ddTTP, ddCTP) 4. The four NTPs 5. A DNA
Polymerase .Primer: A primer is a short nucleic acid sequence which acts as a starting point
for the DNA polymerases enzymes for the complementary strand synthesis. Primer is
generally radiolabelled to generate the autoradiograph. The label may also introduce into
the new strand by adding radiolabelled deoxynucleotides (P32). Template DNA: The ssDNA
to be sequenced is called template DNA. The template DNA is used by DNA polymerase to
attach complementary bases during new DNA synthesis. Polymerase: DNA polymerase is a
type of enzyme that responsible for forming new copies of DNA. In this method DNA
polymerase 1 is used. PROCESS & TECHNIQUE 1. The DNA fragments to be sequenced is
denatured and a single strand of DNA is used as template DNA for replication. 2. This
reaction needs a primer for initiation & usually this is a chemically synthesized
Oligonucleotides. 3. A reaction mixture is prepared by adding Template DNA + Primer + DNA
Polymerase 1 + deoxynucleotide ( dATP ,dGTP,dCTP,dTTP) . 4. The reaction mixture is
divided into 4 tubes with having low concentration of dideoxynucleotide ( dd ATP , dd GTP,
dd CTP , dd TTP) respectively. The dd NTPs act as terminator of chain elongation because the
3' end lacks a hydroxyl group. 5. After suitable incubation Period again heated for the
denaturation of DNA & that samples are electrophoresed in polyacrylamide gel. 6. The
radioactive bands of ss DNA are detected by autoradiography.

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