Higher Biology | Key Area 1.2: DNA Replication

DNA replication

The process by which a cell makes an identical copy of its DNA. Performed prior to cell division so each daughter cell inherits an identical copy of DNA when the cell divides.

What molecules are needed for DNA replication?

- Original DNA template, where each DNA strand can be used as a template to create a new DNA molecule.

- DNA polymerase, an enzyme that adds new nucleotides to a growing strand of DNA using complementary base pairing.

- DNA ligase, which seals fragments of DNA nucleotides together (only in lagging strand).

- Primers, which allows DNA polymerase to add free DNA nucleotides.

- Free DNA nucleotides, to form the new strands.

- ATP, to provide energy for various processes, eg. for helicase to unzip DNA.

Primer

A short strand of nucleotides which binds to the 3’ end of the growing DNA strand, allowing DNA polymerase to bind and add free nucleotides. A primer is therefore required for DNA replication to start, but are eventually replaced by DNA.

DNA polymerase

An enzyme that adds new DNA nucleotides to the deoxyribose 3’ end of a growing DNA strand by complementary base pairing, as it works in a 5’→3’ direction.

Ligase

An enzyme which joins and seals together fragments of DNA nucleotides during the synthesis of the lagging strand.

Give a condensed explanation of the process of DNA replication:

- The DNA double helix structure is unwound and unzipped by the enzyme helicase, which breaks the weak hydrogen bonds between bases (holding strands together) to form two template strands. Process occurs at multiple locations on a DNA molecule.

- A primer attaches to a short sequence of DNA (3’ end), allowing DNA polymerase to bind.

- DNA polymerase adds nucleotides to the new growing deoxyribose 3’ end, in a 5’→3’ direction, using complementary base pairing.

Replication of leading strand

- Primer attaches to short sequence of DNA, allowing DNA polymerase to bind and start replication.

- DNA polymerase adds free DNA nucleotides to the deoxyribose 3’ end of the growing strand, using complementary base pairing.

- Strand synthesised continuously, towards the replication fork.

Replication of lagging strand

- Primer attaches to the end of the other exposed template strand, allowing DNA polymerase to bind and start replication.

- DNA polymerase works in a 5’→3’ direction using complementary base pairing. Since DNA is antiparallel, it adds free nucleotides in fragments to the opposite strand (ie. nucleotides join onto 3’ end of primers and grow the chain).

- Strand synthesised in fragments, away from the replication fork. Fragments of DNA nucleotides are then joined and sealed by the enzyme ligase.

Polymerase Chain Reaction (PCR)

A technique used to amplify DNA in vitro using complementary primers for specific target sequences.

What is required for PCR?

- Sample (template) DNA, ie. the original strand of DNA needing amplified.

- Primers, which join onto the two ends of the region of DNA needing amplified to start DNA synthesis.

- Heat-tolerant Taq DNA polymerase, which adds nucleotides to the growing strand and is not denatured by high temperatures in the reaction.

- Thermal cylinder, which varies the temperature of the reaction.

- pH buffer, which creates the optimum conditions for enzymes.

Function of primers in PCR

Primers are short strands of nucleotides, complementary to specific target sequences at the two ends of the region of DNA to be amplified.

Explain the process of PCR:

- Consists of a series of cycles of repeated heating and cooling, forcing DNA to continually denature, replicate and amplify.

- DNA heated to 92-98°C to break weak hydrogen bonds between bases and separate two strands.

- DNA cooled to 50-65°C to allow primers to bind to target sequences of DNA (attaches at both ends of region to be amplified).

- DNA heated to 70-80°C for heat-tolerant Taq DNA polymerase to replicate the region of DNA by adding free nucleotides at the site of the primer (chain extension).

- Cycle repeated 20-30 times, the sample DNA doubling each time. Yields millions of copies of the original strand of DNA.

Practical applications of PCR

- PCR technique allows specific target sequences of DNA to turn into millions of copies (ie. amplifies tiny amounts of sample) cheaply and easily, so it is used for analysis because it is highly specific and easily automated.

- Settling paternity suits

- Diagnose genetic disorders

- Help solve crimes (forensic evidence)

- Archaeological investigations