Sanger sequencing (Enzymatic DNA sequencing)

Jyoti PawarM.Sc IV sem - Biotechnology

Sanger sequencing Contents

• Introduction of DNA sequencing• History of DNA sequencing• Purpose• Methods of DNA sequencing Maxam & Gilbert method Sanger method• Advantages and Disadvantages• Comparison• Bibliography

1. Introduction of DNA sequencing

The process of figuring out the correct order of the four nitrogen-containing bases:

⁻ Adenine, ⁻ Guanine, ⁻ Cytosine,⁻ Thymine in a section of DNA

“Determining the order of bases in a section of DNA.”

History of DNA sequencing

“DNA sequencing method developed by Fred Sanger “

In the 1980s, two key developments allowed researchers to believe that sequencing the entire genome could be possible.

The first was a technique called polymerase chain reaction (PCR) that enabled many copies of DNA sequence to be quickly and accurately produced.

The second, an automated method of DNA sequencing, built upon the chemistry of PCR and the sequencing process developed by Frederick Sanger in 1977.

Purpose

• Deciphering “code of life”

• Detecting mutations

• Typing microorganisms

• Identifying human halotypes

• Designating polymorphisms

Method of DNA sequencing

To sequence the DNA:• Separated into two strands. • The strand to be sequenced is copied using chemically altered

bases. • These altered bases cause the copying process to stop each

time one particular letter is incorporated into the growing DNA chain.

• This process is carried out for all four bases, and then the fragments are put together like a jigsaw to reveal the sequence of the original piece of DNA.

DNA sequencing Methods

Historically there are two main methods of DNA sequencing:

1. Maxam and Gilbert method2. Sanger method

Modern sequencing equipment uses the principles of the Sanger technique.

MAXAM & GILBERT METHOD

• A. M. Maxam and W.Gilbert-1977

• The sequence of a double-stranded or single-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions.

Principle : MAXAM & GILBERT METHOD

“Chemical degradation”

Reaction in two stages:

• Chemical modification of the bases• Modified base is removed from its sugar, pyperidin cleaves

phosphodiester bonds 5’ and 3’ and base is released.

SANGER METHOD

• Most common approach used to sequencing DNA.

• Invented by Frederick Sanger - 1977

• Nobel prize - 1980

• Also termed as -⁻ chain termination⁻ dideoxy method

⁻ sanger sequencing

Principle: SANGER METHOD

“Method of DNA sequencing “

Based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA

replication.

• The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains.

• These chains terminating at specific nucleotide positions. • Separate by gel electrophoresis• Read DNA sequence

Requirements

DNA sequencing is performed in four separate tubes, each containing

i. Single stranded DNA to be sequencedii. DNA polymeraseiii. Primersiv. The four dNTPs (dATP, dCTP, dTTP and dGTP)v. Small amount of one of the four ddNTPs (ddATP or

ddCTP or ddTTP or ddGTP)

Steps of Dideoxy Sequencing

1. A primer is annealed to a single-stranded section of DNA2. DNA- primer mixture is put into 4 separate tubes with DNA

polymerase and a solution of dNTPs at a concentration of 100 times lower than the dNTP concentration.

3. DNA Pol uses dNTPs to extend the DNA4. ddNTPs are put together randomly, resulting in different lengths of

fragments5. Fragments that are from each of the reactions are denatured and

separated by size using gel electrophroesis6. The gel is used to visually detect the DNA fragments. The fragments

are to be read from bottom to top and this represents the complementary sequence of the original strand of DNA.

Advantages and Disadvantages

• Allows scientists to determine genome sequence

• Identify the genes causing genetic diseases

• Provides details about individuals and their families

• Police have DNA samples of people that have committed no crime

COMPARISON

• Sanger Method• Enzymatic• Requires DNA synthesis• Termination of chain

elongation• Automation

• Single-stranded DNA

• Maxam Gilbert Method• Chemical• Requires DNA• Breaks DNA at different

nucleotides• Automation is not

available• Double-stranded or

single-stranded DNA

Bibliography

• "Dideoxy Sequencing Animation." Dideoxy Sequencing Animation. Web. 23 May 2012.

Link <http://spine.rutgers.edu/cellbio/assets/flash/dideoxy.htm>.

• "Sanger's Method of Dna Sequencing Video." World News. Web. 23 May 2012.

Link <http://wn.com/Sanger's_Method_of_DNA_Sequencing_Video>.

• "DNA Sequencing: Method, Benefits and Applications." DNA Sequencing: Method, Benefits and Applications. Web. 23 May 2012.

Link <http://www.biotecharticles.com/Genetics-Article/DNA-Sequencing-Method-Benefits-and-Applications-248.html>.

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