24. Double stranded DNA replicates in a semi-conservative manner. In an in vitro DNA synthesis reaction, dideoxyCTP and, dideoxy CMP were individually added in excess (in separate reaction tubes) in addition to dNTPs

and other necessary reagents. Rate of DNA Synthesis was measured by incorporation of 3H-thymidine. The four graphs drawn below represent the rate of DNA synthesis in two separate reaction tubes.
Which of the following graphs represents the expected data?

Introduction

DNA synthesis by DNA polymerases requires the incorporation of deoxyribonucleoside triphosphates (dNTPs) into a growing DNA strand. Dideoxynucleotides (ddNTPs), lacking a 3′-OH group, act as chain terminators because their incorporation prevents further nucleotide addition. This property is exploited in Sanger sequencing to generate DNA fragments of varying lengths.

In an in vitro DNA synthesis assay containing DNA polymerase, Mg^2+, dATP, dGTP, dCTP, dTTP (including ^3H-thymidine for detection), the addition of ddCTP or dideoxy CMP (ddCMP) separately influences DNA synthesis differently:

• ddCTP (dideoxycytidine triphosphate): A chain-terminating nucleotide analog that competes with dCTP for incorporation. Once incorporated, it halts DNA synthesis immediately.

• ddCMP (dideoxycytidine monophosphate): The monophosphate form lacks the triphosphate group necessary for incorporation by DNA polymerase and thus does not act as a chain terminator.

Expected Effects on DNA Synthesis Rate

1. With ddCTP added in excess:

   • DNA polymerase incorporates ddCTP in place of dCTP randomly during DNA synthesis.

   • Incorporation of ddCTP terminates chain elongation because it lacks the 3′-OH group required for phosphodiester bond formation with the next nucleotide.

   • This causes a sharp decrease in the rate of DNA synthesis as more chains terminate prematurely.

   • The DNA synthesis curve will show a rapid decline compared to control without ddCTP.

2. With ddCMP added in excess:

   • ddCMP cannot be incorporated into DNA because DNA polymerase requires nucleoside triphosphates (dNTPs) for polymerization.

   • ddCMP acts as an inert molecule in this context and does not inhibit DNA synthesis.

   • The rate of DNA synthesis remains similar to the control reaction with only dNTPs.

Summary of Expected Graph Patterns

Explanation

• Dideoxynucleotides (ddNTPs) lack the 3′-OH group essential for DNA chain elongation, causing termination upon incorporation.

• Dideoxy nucleoside monophosphates (ddNMPs) cannot be incorporated by DNA polymerase as they lack the triphosphate group necessary for catalysis.

• Therefore, only ddNTPs act as effective chain terminators and inhibitors of DNA synthesis.

Conclusion

In an in vitro DNA synthesis reaction, the addition of ddCTP results in a significant decrease in DNA synthesis rate due to chain termination. In contrast, ddCMP does not inhibit DNA synthesis because it cannot be incorporated by DNA polymerase. The expected graph will show a sharp decline in synthesis rate with ddCTP, while the rate remains unchanged with ddCMP.