1.2 - Replication of DNA

Question 1

DNA Replication

Answer 1

DNA replication is the process by which a cell makes an identical copy of its DNA.

This process is performed at the beginning of/prior to every cell division so that when the cell divides, each daughter cell will inherit an identical copy of the DNA.

Question 2

Requirements for Replication of DNA

Answer 2

DNA Nucleotides - Added to form the two new strands of DNA

Two template strands - To allow DNA to be copied onto

DNA polymerase - An enzyme that replicates DNA. Adds DNA nucleotides to the deoxyribose sugar at the 3’ end of the new DNA strand which is forming.

Ligase - An enzyme that joins fragments of DNA nucleotides together.

Primers - Starts DNA replication. A primer is a short strand of DNA nucleotides which binds to the 3’ end of the templet strand. This allows polymerase to add DNA nucleotides.

Question 3

DNA polymerase can only add complementary…

Directionality of DNA polymerase

Answer 3

DNA polymerase can only add complementary nucleotides onto the 3’ end. This means that one leading strand is replicated continuously whilst the other lagging strand is replicated in fragments.

Question 4

Process of DNA Replication (summarised)

Answer 4

1. DNA is unwound and the hydrogen bonds between the bases are broken - forming 2 template strands
2. Primers are attached to a short sequence of DNA allowing DNA polymerase to bind
3. DNA polymerase will add nucleotides using the complementary base pairing rule to the deoxyribose (3’) end of the NEW strand which is forming.

Question 5

Leading Strand

Answer 5

Step 1 - DNA is unwound and unzipped. This breaks the hydrogen bond between complementary bases to form the template strands.

Step 2 - Primers are attached to the 3’ end of the leading strand to allow replication to start.

Step 3 - DNA polymerase adds DNA nucleotides, using complementary base pairing, onto the deoxyribose sugar 3’ end of the template DNA strand.

Question 6

Lagging Strand

Answer 6

Step 1 - Many primers are used on the lagging strand at various 5’ ends as the strand is exposed.

Step 2 - DNA polymerase adds DNA nucleotides onto the deoxyribose sugar 3’ end in fragments.

Step 3 - Ligase joins the fragments of DNA.

Question 7

Polymerase Chain Reaction (PCR)

Answer 7

PCR is a technique for the amplification of DNA in vitro (outside of a cell).

PCR amplifies DNA using complementary primers for specific target DNA sequences.

Question 8

Thermocycler

Answer 8

A thermocycler is the equipment used in the process of PCR. It varies the temperature of the reaction.

Question 9

Requirements for PCR

Answer 9

• DNA nucleotides – the original strand of DNA which needs amplified.
• Primers - primers are short complementary sequences of nucleotides needed to start DNA synthesis.
• pH buffer -
• DNA template strand - to synthesise the new strands of DNA.
• Heat-tolerant DNA polymerase - an enzyme which will add nucleotides to the growing strand and which is not denatured by the high temperatures used in the reaction.

Question 10

PCR and Primers

Answer 10

In PCR, primers are short strands of nucleotides which are complementary to specific target sequences. They attach at the 2 ends of the region of DNA to be amplified.

Question 11

Uses of PCR

Answer 11

• Solving crimes
• Settling paternity suits
• Diagnostic genetic disorders

Question 12

Stages of PCR

Answer 12

Stage 1: 92°C to 98°C - DNA is heated to break the hydrogen bonds/denature the DNA and separate the two strands.

Stage 2: 50°C to 60°C - DNA is cooled to allow primers to bind to their complementary target sequences.

Stage 3: 70°C to 80°C - DNA is heated for the heat tolerant DNA polymerase to replicate the region of DNA. Two strands are formed

This would be just one cycle, so a single sample of one DNA molecule is amplified into two. This cycle is repeated 20-30 times, yielding millions of copies of the original sample of DNA.

Question 13

Gel Electrophoresis

Answer 13

Gel electrophoresis is a technique used to separate DNA strands of different lengths.
The DNA is placed in small wells at the top of the an agarose gel, and an electric field applied.

The current is switched on and the DNA is attracted to the positive electrode due to its negative charge.

The longer the pieces of DNA, the harder it is for it to move through the gel - so the larger fragments are found nearer the wells, whilst the smaller fragments move faster and are located nearer the positive electrode at the opposite side from the wells.

The DNA must be stained before it can be visualised in the gel.