Sanger sequencing

Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

1. Sanger sequencing (Enzymatic DNA sequencing) Jyoti Pawar M.Sc IV sem - Biotechnology

2. 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

3. 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.”

4. 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 .

5. Purpose Deciphering “code of life” Detecting mutations Typing microorganisms Identifying human halotypes Designating polymorphisms

6. 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.

7. DNA sequencing Methods Historically there are two main methods of DNA sequencing: Maxam and Gilbert method Sanger method Modern sequencing equipment uses the principles of the Sanger technique.

8. 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.

9. 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.

10. 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

11. 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

12. Requirements DNA sequencing is performed in four separate tubes, each containing i .   Single stranded DNA to be sequenced ii.  DNA polymerase iii.  Primers iv.  The four dNTPs (dATP, dCTP, dTTP and dGTP) v.   Small amount of one of the four ddNTPs ( ddATP or ddCTP or ddTTP or ddGTP )

13. Steps of Dideoxy Sequencing 1. A primer is annealed to a single-stranded section of DNA 2. 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 DNA 4. ddNTPs are put together randomly, resulting in different lengths of fragments 5. Fragments that are from each of the reactions are denatured and separated by size using gel electrophroesis 6. 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.

14. 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

15. 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

16. 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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