B: Principles of Nucleic Acid Hybridisation

Question 1

–List three ways that DNA can be denatured, name the enzyme that unwinds DNA.

Answer 1

• Heat, high pH, enzymes, physically
• Helicase unwinds DNA

Question 2

–Explain what is meant by DNA melting temperature, what is the approximate Tm of GGGAAATTTCCC?

Answer 2

• It is the temperature at which two DNA strands will seperate because of the heat breaking the H bonds binding the base pairs together. A-T bonds break at 2 degrees and G-C bonds break at 4 degrees.
• Tm = 36 degrees

Question 3

–Write out a definition of DNA annealing and DNA hybridisation.

Answer 3

DNA annealing/hybridisation is when two single strands of complementary DNA are free in a solution, and they find and base pair with each other a duplex (one double strand of DNA)

Question 4

—Explain how and why a DNA probe can be used to find its complement in a separate DNA molecule.

Answer 4

• How: If a 20 base oligonucleotide is made that is complementary to a 20 base sequence in the genome, it should hybridise specifically to that sequence and not to any other sequence. So, one piece of DNA (DNA probe) can be used to find its specific complement in a different piece of DNA.
• Why: DNA probes can be used to identify the presence or absence of a certain sequence in a genome, such as a mutation.

Question 5

—Explain how DNA probes and hybridisation can be used to detect the normal and commonly mutated allele (delta F508) of the CFTR gene in a DNA sample from an individual.

Answer 5

DNA probe 1: matches normal gene
- DNA probe is fluorescently labelled
- Probe matches normal allele at 25 degrees - 26 degrees is melting temperature.
- Melting temperature for probe to bind to mutant gene is only 12 degrees because it does not line up with mutant gene.

DNA probe 2: matches mutant gene
- Binds to mutant at 24 degrees
- Binds to normal at 22 degrees.