Explain DNA replication as semi-conservative using evidence from the Messelson-Stahl experiment.
Both parental strands contained only heavy 15N stands.
Each parental 15N strand served as the template for replicating a new 14N strand.
In the first generation, the isolated DNA would show only one single band of intermediate density where each replicated hybrid DNA molecule was composed of one new 14N strand and one parental 15N strand => semi-conservative (each daughter molecule consists of 1 original strand and 1 newly synthesized strand)
- Helicase
- unwinds the double helix by breaking the hydrogen bonds between the complementary base pairs in the parental strands.
- Without helicase, the double-stranded DNA would not unwind
- Primase
- attaches to the unwound chain and catalyses the synthesis of RNA primer to provide free 3’OH ends for DNA polymerase III
- Without Primase, no RNA primers will be synthesised / No free 3’OH ends will be available for adding deoxyribonucleotides
- DNA polymerase III
elongates the new daughter strand in the 5’ to 3’ direction by catalysing the phosphodiester bond formation between the incoming deoxyribonucleotides and the free 3’OH end of the daughter strand
- Free deoxyribonucleotides
are incorporated by complementary base pairing to the parental DNA strands, with adenine to thymine and guanine to cytosine
- Leading vs lagging strand
- In a replication fork, the leading strand is synthesised continuously (leading strand is the strand that can form in the 5’ to 3’ direction smoothly) while lagging strand is synthesised discontinuously to form Okazaki fragments
- RNA primers
are removed and replaced by deoxyribonucleotides by DNA polymerase I
- Nicks between the Okazaki fragments
- are filled in by DNA ligase by forming phosphodiester bonds between Okazaki fragments
- without DNA ligase, daughter strand will be incomplete due to presence of nicks between Okazaki fragments which are not filled in
Gel electrophoresis
a technique used to separate DNA, RNA and proteins that differ in size, charge or conformation
Explain how gel electrophoresis is used to analyse DNA, including how the DNA fragments on a gel are made visible and the role of DNA markers in identifying DNA fragments of different size.
- DNA has a negative charge and will migrate towards the anode under an electric current.
- The agarose gel acts as a molecular sieve to separate DNA based on their size.
- DNA fragments which are longer/with heavier molecular weight will migrate slower and hence travel a shorter distance than those which are shorter/with lighter molecular weight
- Nucleic acid dye is used to stain the agarose gel before or after electrophoresis to visualise DNA in gel electrophoresis → Nucleic acid dyes bind to DNA to form a complex, which can then be visualised by exposing the gel to UV or blue light
- DNA markers are used to identify DNA fragments of different size
