Question

The Sanger method of DNA sequencing requires what type of dNTP modification to generate truncated DNA products? How are individual dNTPs identified? How are the different sizes of DNA separated from each other?

Answer 1

Sanger sequencing is a method of DNA sequencing. This method is generally used to sequence up-to 800 base pairs. Sanger sequencing applies the principle of chain termination where modified deoxynucleotide triphosphates (dNTPs) are used which are called 2’,3’-dideoxynucleotides triphosphates (ddNTPs). A nucleotide triphosphate is made up of deoxyribose sugar, nitrogenous base attached at the 1’ position of the sugar and a phosphate group at the 5’ position. When the nucleotide attaches to a growing chain of nucleotides forming a DNA strand, the phosphate group of the nucleotide attaches to the 3’ position of the previous nucleotide’s deoxyribose sugar. This is because of the hydroxyl group (-OH) present at the 3’ position in the sugar to which the phosphate group attaches via phosphodiester bond. But in case of a ddNTP, the 3’ position lacks the -OH group. So the incoming nucleotide cannot form the phosphodiester bond and the chain terminates at the dideoxynucleotide. Due to this, when dideoxy nucleotides are included in the reaction, DNA strands of varying lengths are recovered. Sanger sequencing is therefore also called chain terminating or dideoxynucleotide sequencing.

An essential required for DNA sequencing is single stranded DNA (ssDNA) (template DNA) which is obtained by polymerase chain reaction (PCR). The ssDNA obtained is mixed with the following components to prepare an aliquot: oligonucleotide primer, DNA polymerase enzyme, the deoxynucleotide triphosphate (either of dATP, dCTP, dGTP, and dTTP in each aliquot). Each aliquot with the specific dNTP is added with its respective ddNTPs (ddATP, ddCTP, ddGTP, or ddTTP) which is radioactively labelled with isotope ³²P. Apart from radioactive labelling, fluorescent labelling of the ddNTPs is also favoured where each of the four ddNTPs are labelled with different fluorescent dyes.

At the 3’ end of the DNA strand, the primer attaches and begins the complementary strand synthesis. Since the ddNTPs terminate the chain formation after incorporation in the growing DNA strand, various DNA strands with different lengths but same 3’ end will be obtained for each ddNTP.

To identify the dNTPs in the DNA chain before it terminated by the addition of ddNTPs, the aliquot for each dNTP is loaded in individual wells in a thin, long polyacrylamide gel. Since the ddNTPs are radioactively labelled, they can be analysed through autoradiography. Each ddNTP in the gel will appear as a dark band and the position of each band in a lane specifies the position of the nucleotide in the DNA strand. In fluorescent labelling, the color of the nucleotide bands is considered while sequencing. The bands are read from bottom to top of the gel. The sequence obtained is in 5’-3’ direction. This is the sequence of the strand synthesized from the original template DNA. The complementary sequence of the synthesized DNA strand will be the template DNA sequence.