Question

  Which of the following is/are positively correlated to the melting temperature (Tm) of a DNA sequence? (1pt)

A. Its annealing temperature

B. Its GC content

C. The number of hydrogen bounds it can form with a template

D. The length of the sequence

E. All of the above

When comparing CRISPR as an immune system to CRISPR as a molecular technique, which is the equivalent of the repeat sequence found in the bacterial genome? (1pt)

A. The spacer sequence of the gRNA

B. The PAM site

C. The scaffold sequence of the gRNA

D. None of the above

1. To improve a PCR protocol, a solution of 1.2% MgCl2 needs to be added to the PCR reaction. What volume of MgCl2 is necessary so its final concentration equals 1.5mM? (2pts)

Molar mass of MgCl2 = 96g/mol

Final volume of the PCR reaction: 50µL

Answer 1

Ans 1: Its GC content, the number of hydrogen bonds it can form template with and the length of the sequence.

Melting temperature of a DNA is defined as the temperature at which 50% of the double stranded DNA (dsDNA) has been denatured to single stranded DNA (ssDNA). The melting temperature of a dsDNA depends on its GC content because Guanine forms base pair with Cytosine with 3 hydrogen bonds where as Adenine forms base pair with Thymine with 2 hydrogen bonds. Thus higher the no. of GC base pair, greater is the no. of hydrogen bonds present hence higher temperature is required to melt the dsDNA. The melting temperature of a dsDNA increases with increase in the no. of hydrogen bonds since more amount of energy in the form of heat is required to break the hydrogen bonds and melt the dsDNA. Also, as the length of the dsDNA sequence increases, the no. of H bonds also increases significantly thereby increasing the melting temperature. Option A is not correct because annealing is defined as the process where the dsDNA is separated and temperature is lowered during a PCR reaction where the primers bind to the ssDNA strand.