Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)

Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.

1. DNA Sequencing ENZYMATIC METHOD Presented by, Raihanathus Sahdhiyya A, I M.Sc. Microbiology

2. What is DNA sequencing? Process of determining the sequence of nucleotides Adenine Thymine Cytosine Guanine in a piece of DNA.

3. Sanger sequencing Fredrick Sanger with his colleagues developed this method of sequencing in the year 1977 , for which he was awarded Nobel in 1980 It is also known as Chain termination method, Dideoxy sequencing or Enzymatic method It was the most widely used DNA sequencing method for almost 40 years, bringing successful completion of the Human Genome Project (HGP) in 2003 Sanger sequencing method is based on the chain termination by the use of Dideoxynucleotides (ddNTPs)

4. What is Dideoxynucleotide ? (ddNTPs) A  dideoxynucleotide  (ddNTP) is an artificial molecule that lacks a hydroxyl group at the 3' carbon of the ribose sugar

5. Sanger sequencing

6. Sanger sequencing process

7. The first step is to fragment the DNA by applying heat and clone the fragments into vectors 1. DENATURE & CLONE

8. The second step is to anneal a synthetic oligonucleotide The oligonucleotide acts as a binding site for a primer and provides a 3' hydroxyl group, which is necessary to initiate DNA synthesis In order to recognize the sequence and identify precisely the first nucleotide of the target DNA, the primer is usually positioned 10 to 20 nucleotides away from the target DNA. 2. PRIMER ATTACHMENT

9. Four different reaction vials are made, each with the four standard dNTP's, and DNA polymerases The difference among the vials are the type of ddNTPs. Each vial will have 1 ddNTP per 100 dNTP 3. ADDITION OF dNTP s AND ddNTPs

10. After DNA synthesis occurs, each reaction vial will have a unique set of single-stranded DNA molecules of varying lengths. However, all DNA molecules will have the same primer sequence at its 5' end The resulting DNA fragments are then denatured by heat since base-paired loops of ssDNA may cause difficulty in resolving bands when running a gel. Additionally, formamide may also be added to prevent base pairing. (contd.)

11. As the sequences vary in size, they have to be lined up according to the size to determine the sequence 4. SEQUENCING BY GEL ELECTROPHORESIS Radiography Here, the ddNTPs would have to be radioactively or fluorescently labeled beforehand for automated sequencing machines. The DNA strands are then separated using gel electrophoresis, then read from top to bottom (3' to 5') to obtain the sequence.

12. Dye-terminating sequencing Each ddNTP is fluorescently labeled to use dye-terminating sequencing. This causes each of the four ddNTPs to emit light at different wavelengths. Here, capillary electrophoresis is carried out, with a single lane to capture the nucleotide sequence. (contd.)

13. Not as toxic and less radioactivity than Maxam and Gilbert method Easier to automate - Leroy Hood and coworkers used fluorescently labeled ddNTPs and primers for the first high-throughput DNA sequencing machine. This lowered the cost from $100 million to $10,000 USD in 2011 Highly accurate long sequence reads of about 700 base pairs Easier to get started. The kits that are commercially available contain reagents necessary for sequencing - pre- aliquoted and ready to use. ADVANTAGES

14. Poor quality in the first 15-40 bases of the sequence. This is due to primer binding and deteriorating quality of sequencing traces after 700-900 bases Time consuming, especially due to requirement for electrophoretic separation of fragments DNA fragments cloned before sequencing - read may include parts of the cloning vector Only short 300-1000 nucleotides long DNA fragments in a single reaction. Problem with reading strands longer is the insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide. DISADVANTAGES