Question

In one reaction used for DNA sequencing, 2,3 dideoxycytosine triphosphate is added to the reaction mixture. Why?

-It is used to fluorescently mark cytosine in the DNA chain.
-It is used as a primer to start synthesis at positions along the DNA that complement cytosine.
-It is used to terminate DNA chain growth at sites where cytosine occurs.
-It is used to stimulate the polymerase chain reaction and allow the template to be copied.
-It is used to bind to and separate the DNA pieces on the gel.

Answer 1

2,3 dideoxycytosine triphosphate (ddCTP) is used in DNA sequencing to terminate DNA chain growth at cytosine sites, preventing further elongation and enabling sequence determination via gel electrophoresis and laser scanning.

Step-by-step explanation:

In DNA sequencing, especially the method known as Sanger sequencing, 2,3 dideoxycytosine triphosphate (ddCTP) is used to terminiate DNA chain growth at sites where cytosine occurs. The ddCTP lacks a 3'-OH group, which is necessary for forming a phosphodiester bond with the next nucleotide. Hence, when it is incorporated into the DNA chain, it prevents further elongation. Each of the four different dideoxynucelotides (ddNTPs) used in separate reactions is labeled with a different fluorescent dye, which allows for the detection of each nucleotide in the sequence during analysis by gel electrophoresis and laser scanning.

DNA polymerases normally add nucleotides to a growing DNA strand by creating a bond between the phosphate of a nucleotide triphosphate and the free 3' OH group of the deoxynucleotide at the end of the strand. However, ddNTPs such as ddCTP are designed without the critical 3' OH, inhibiting further chain growth. This controlled interruption of synthesis enables the determination of the DNA sequence when a mixture of normal nucleotides and limited amounts of ddNTPs are added. Thus, by causing selective chain termination, ddNTPs play a crucial role in the DNA sequencing process.