1.2 - Replication Of DNA Flashcards
Requirements for DNA replication
- DNA to act as a template
- Primers
- Nucleotides
- DNA polymerase (enzyme)
- Ligase (enzyme)
- ATP
Stage 1 of DNA replication (replication fork)
The DNA molecule unwinds, and weak hydrogen bonds between base pairs break allowing the two strands to separate (unzip). This form a replication fork.
Stage 2 of DNA replication (primer)
DNA polymerase can only add nucleotides to the 3’ end of an existing chain. A short sequence of nucleotides called a primer is needed. It attaches to the 3’ end of the parent strand to be replicated.
Stage 3 of DNA replication (leading strand)
DNA polymerase now adds free complementary nucleotides to the 3’ end of the primer and the leading strand forms continuously. This is called the leading strand.
Stage 4 of DNA replication (lagging strand + ligase)
DNA polymerase can only add to the 3’ end of a strand. Therefore the opposite (lagging) strand is replicated in fragments as the DNA unzips.
The enzyme ligase joins the fragments together on the lagging strand.
Requirements of PCR
- Primers
- DNA molecule to be copied
- DNA polymerase
- DNA nucleotides
Step 1 of PCR (95C)
Double stranded fragment of DNA to be amplified is heated (95C) to break the hydrogen bonds between bases and separate the two strands in the double helix.
Step 2 of PCR (55C)
The now separated DNA strands are cooled to approx 55C. This allows the primers in the mix to bind to their complementary target strand.
Step 3 of PCR (72C)
The mixture is then incubated with the heat tolerant DNA polymerase and the four bases (A T C G) at approx 72C
Diagnostic application of PCR
Used diagnostically to diagnose genetic diseases and to detect low levels of viral infection.
Forensic application of PCR
Used in forensic medicine to analyse minute traces of blood and other tissues (as little as a single cell)
