Question

Describe the purpose of adding both dNTPs and ddNTPs to the reaction mix when conducting dideoxy chain-termination DNA sequencing. Why would one want a greater abundance of dNTPs in the reaction mix than ddNTPs?

Answer 1

Answer-

1. In Sanger's method of dideoxy chain termination dideoxy nucleotides are used to terminate replication during polymerase chain reaction. The dNTPs are used for polymerization of new DNA strand and ddNTP is used to terminate synthesis. As ddNTP lacks 3'-OH it can not form phosphodiester bond with next nucleotide and hence chain gets terminated whenever ddNTP incorporated into new DNA strand.

2. To sequence DNA, primers are used to provide substrate for DNA polymerase and nucleotides are added complementary to parent DNA strand. But to sequence parent DNA, polymerization should be stop at each base after primer stepwise and this is done by dideoxy nucleotide that lacks 2'and 3'-OH of ribose sugar. If we use large amounts of ddNTP then chain termination occurs near to primer and we will get shorter products. So that complete DNA sequence can not be identified as chain gets terminated earlier.

3. To avoid this problem while setting reaction mixture all four NTPs with small concentration of only one type of ddNTP is added so that chain termination will not occur frequently and we will get different sized products from smaller to longer by which sequence can be determined .