Answer to Question #157110 in Biochemistry for Amelia

Question

Deoxyribonucleotide triphosphates (dNTPs: dATP, dTTP, dCTP and dGTP) are critical components in polymerase chain reactions (PCR) for the amplification of DNA fragments in the lab. Each dNTP is provided individually as a stock solution with a concentration 1 mM.

a)  What volume of each of the four dNTP stock solutions, and what volume of water must be mixed together to prepare 200 μL of solution A, in which the concentration of each dNTP is 0.1 mM (i.e. in solution A the concentration of the four dNTPs will be 0.1 mM dATP, 0.1 mM dTTP, 0.1 mM dCTP, 0.1 mM dGTP)? (3 points)

b)  0.1 μL of solution A were added in a PCR reaction with a final volume of 20 μL. What is the concentration of each individual dNTP in the PCR reaction? Give your answer in units of nM. (3 points)

Answer 1

A PCR template for replication can be of any DNA source, such as genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA. Nevertheless, the composition or complexity of the DNA contributes to optimal input amounts for PCR amplification. For example, 0.1–1 ng of plasmid DNA is sufficient, while 5–50 ng of gDNA may be required as a starting amount in a 50 µL PCR. Optimal template amounts can also vary based on the type of DNA polymerase used; a DNA polymerase engineered to have higher sensitivity due to affinity to the template would require less input DNA. Optimization of DNA input is important because higher amounts increase the risk of nonspecific amplification whereas lower amounts reduce yields