D.1.1 - DNA Replication

Why does the production of DNA need to be accurate?

So new cells/organisms can carry out functions of life

What does DNA replication do?

Doubles the quantity of DNA

What is achieved by DNA replication?

Reproduction, growth & repair, tissue replacement

Introducing new bases to DNA

An experiment was done, introducing two new nitrogenous bases to the genetic code of bacteria. These bases were incorporated into the bacterial DNA code. This could be the basis for new medicines.

Why is it called semi-conservative replication?

• Each strand in the DNA double helix acts as a template for the synthesis of a complementary strand

• Each daughter DNA strand contains one old strand from the parental DNA and one new one

• Hence the name semi-conservative

Helicase

The enzyme that unzips/unwinds the DNA double helix in the first step of DNA replication, breaks the hydrogen bonds joining organic bases.

DNA polymerase

Group of enzymes that join free-floating nucleotides onto single DNA strands to synthesise a parallel DNA double strand. (Uses the single DNA strand as a template), catalyses hydrogen bonds between the organic bases. Can only synthesise from 5’ to 3’ end.

Why are daughter strands from the same DNA strand synthesised in opposite directions?

Because DNA polymerase can only synthesise from the 5’ end (of the new strand) to the 3’ end. These are opposite in each strand because of DNA’s antiparallel structure (the two strands run in opposite directions.)

Outline DNA replication

• DNA is unzipped by helicase

• Two identical, complementary strands are formed

• Free-floating nucleotides in the nucleoplasm join with the now-unpaired organic bases in the DNA single strand

• These nucleotides form covalent bonds, catalysed by one of the DNA polymerase enzymes, which make up the DNA backbone

• The two new DNA strands are synthesised in opposite directions, one towards the helicase, and one away from it.

Summarise semi-conservative DNA replication

Helicase enzyme unzips a DNA double helix strand, allowing for two new, identical strands to be synthesised. Free-floating nucleotides are joined to the single strands by DNA polymerase enzymes

Why are the two daughter DNA strands synthesised from one DNA strand identical?

Because of the base pair rule

Replication fork

The shape of DNA during step one of replication: the double helix on one side of the moving helicase, the other side already separated by helicase, leaving 2 separated strands.

PCR

A thermocycler is used to take a small amount of DNA, then it copies all the nucleotides and can produce millions of copies of the DNA. PCR can only amplify a targeted section of DNA.

What is needed for PCR?

• Primers

• Taq polymerase (enzyme)

• Free nucleotides

• Helicase

PCR primers

Short, single-stranded polymers of nucleotides that are complementary to the nucleotides at one end of the target DNA. Neccessity: DNA polymerases can only attach new DNA nucleotides to existing polymers.

PCR process

1. Denaturation: 92-98°C, mixture is heated to break hydrogen bonds holding together the two DNA strands

2. Annealing: 50-65°C, allows primers to bind with nucleotides at the ends of target DNA

3. Elongation: 70-80°C, taq polymerase catalyses DNA replication by extending the primers.

Gel electrophoresis

Enzymes are used to fracture long DNA filaments into smaller particles of varying size. They are then placed in the wells of an electrophoresis chamber and exposed to an electric current. The largest, heaviest, and least charged particles move with great difficulty, while the smallest, least massive, and highly charged move with ease. This difference in speeds creates a banded pattern of DNA.

DNA profiling/fingerprinting

The process of comparing two different DNA samples to see if they correspond. Done using gel electrophoresis; if the patterns of DNA are identical, the DNA belongs to the same individual, if they are similar, the individuals are likely related. When little DNA is present, PCR is used to amplify the amount available for sampling.

Is DNA positively or negatively charged?

Negatively charged due to the phosphates

When is PCR used?

• GMOs

• Criminal investigations

• Paternity testing

• Testing for viral infection (PCR test)

• Genetic diseases

Short tandem repeats

Repeated sequence of A-G-A-T, amount of repetitions varies per person. These create the band patterns made by gel electrophoresis.