Question

1. Which would have the highest melting temperature?

A. TTAACCGGGCTA

B. CATAACAAATCA

C. CGCAATAATACA

D. CGTAATAATACA

D. CGCAACCATCCG

2. Select the ones that can be applied to PCR, there can be more than one right answer.

A. PCR can be used to obtain large quantities of a particular sequence of DNA

B. It can use short synthetic oligonucleotide primers

C. Fe2+ ions are needed for the DNA polymerase.

D. DNA polymerase can be used to synthesize DNA

E. Primers can be used and they are complementary to each other

Answer 1

1. The higher the GC content of a DNA strand, the higher will be its melting temperature as repeating G and C residues can form intermolecular hydrogen bonds in a single stranded DNA strand. GC base pair is linked by three hydrogen bonds and is more stronger than an AT base pair, which is linked by two hydrogen bonds. It is calculated by the formula:

GC content (%) = ( Number of G,C bases/ total number of bases) X 100

The GC content of DNA sequences denoted in option A, B, C, D and E are  50, 25, 33.3, 25 and 66.7% respectively. Thus E will have the highest melting temperature.

2. PCR or polymerase chain reaction is molecular biology technique used to amplify or produce large number of copies of a template DNA. It is achieved by using a pair of short oligonucleotides or primers (that are complementary to and flank the DNA template to be amplified), a thermostable DNA polymerase. Each cycle of PCR consists of three steps: Denaturation, in which the template DNA is heated to a high temperature (95⁰C) in order to separate the two strands apart. This is followed by annealing stage, in which the temperature is lowered down to (50-65⁰C) in order to allow the primers to anneal to the their complementary sequence on template DNA. The annealed primers are then extended in the Extension step by a thermostable DNA polymerase that adds complementary dNTPS in the 5` to 3` direction. The DNA template is doubled after each cycle.

Thus statements A and B are true for PCR. Whereas statement C, D and E are false as Mg²⁺ (and not Fe ²⁺) ions acts as cofactor for Taq polymerase, Taq polymerases are mostly used for PCR reactions as they can withstand the high temperatures used in Denaturion step and normal DNA polymerases cannot. Lastly, primers are complemntary to the template DNA and not to each other.