Molecular Genetics

Question 1

Describe the process of DNA replication.

Answer 1

Both strands of a DNA molecule
separate, each strand acting as templates for the synthesis of two new daughter strands.

The sequence of DNA on the newly
synthesised strands is determined by
complementary base pairing.

New nucleotides complementary-base pair with nucleotides on template strand. The bases are held in place by hydrogen bonding.

DNA polymerase catalyses the formation of phosphodiester bond between adjacent nucleotides to connect them into a strand.

Question 2

Describe the process of transcription.

Answer 2

The enzyme RNA polymerase attaches
to DNA and starts to unwind and separate the DNA double helix. One DNA strand acts as template.

RNA nucleotides base-pair one by one with DNA bases on the template DNA strand via complementary base pairing.

RNA polymerase connects RNA nucleotides into a polynucleotide chain by catalyzing the formation of phosphodiester bonds

As RNA polymerase moves along the DNA, it unwinds and exposes more of the template DNA strand for base pairing with RNA nucleotides.

At end of transcription, RNA polymerase and mRNA released from DNA and the DNA rewinds into a double helix.

Question 3

Describe the process of translation.

Answer 3

In a ribosome, the anticodon of each tRNA complementary-base pairs with a codon of the mRNA.

The ribosome catalyses the formation of peptide bonds between two amino acids that are attached to the two tRNAs currently attached to the mRNA.

The unbound tRNA leaves and another tRNA arrives. The ribosome moves along the mRNA by one codon to repeat the process.

When the ribosome reaches the stop codon, translation stops.

Question 4

Why are bacterial plasmids used as DNA cloning vectors?

Answer 4

• Can easily incorporate foreign DNA with MCS that contains unique restriction sites to insert target DNA.
• Readily taken up by bacteria
• Often contains genes coding for antibiotic resistance, allowing for easy identification of transformed bacteria during selection
• Can self-replicate with origin of replication (ORI) gene and so is present in one or more copies in one bacterium, resulting in greater yield of target DNA.

Question 5

How do restriction enzymes function?

Answer 5

• Each restriction enzyme only recognises a specific DNA sequence (restriction site) that is palindromic.
• Enzyme cleaves the sugar-phosphate backbone of DNA by catalysing breaking down of phosphodiester bonds.
• Same enzyme used to cut gene of interest and plasmid, produce complementary sticky ends
• Restriction site sequence of restriction enzyme should not be found within target DNA’s sequence. (restriction sites should be flanking left and right of target DNA)

Question 6

Describe the function of DNA Ligase.

Answer 6

• The complementary sticky ends of the cut plasmid and gene of interest are held together by weak hydrogen bonds to form a recombinant plasmid.
• DNA ligase is then used to repair the sugar phosphate backbone by catalysing the formation of phosphodiester bonds to form an unbroken recombinant plasmid with an intact sugar-phosphate backbone.

Question 7

Describe transformation and selection.

Answer 7

Transformation:
Recombinant plasmid introduced into the bacteria (with heat shock)

Selection:

• Grow the bacteria on agar medium containing the antibiotic
• Transformed bacteria will survive and form colonies
• Non-transformed bacteria will be killed by antibiotic

Grow bacteria colonies that survived in a growth medium.