D1.1 DNA replication Flashcards
Semi-conservative nature of DNA replication
- Each strand in double helix acts a template for the synthesis of a new, complementary strand
- Each daughter DNA contains an old strand from the parental DNA double helix and a new strand
Meselson & Stahl experiment
1. culture E. coli in a medium with 15N to integrate 15N into bacterial DNA.
2. later, they revised the medium to normal 14N medium.
3. they took samples and checked for the density of DNA regularly
2 types of molecules needed For DNA rep
- Enzymes
- Free nucleotides
Process of DNA rep
- Helicase breaks the hydrogen bonds between DNA double helix, unzipping it into 2 strands. Binding proteins stabilise the 2 strands, preventing them from winding up again.
- The point at which helicase is working to unzip the DNA molecule is known as replication fork
- On each strand, DNA primase creates an RNA primer that binds to the DNA strand. Several RNA primers are needed for the lagging strand.
- Free nucleotides in the nucleus floats to their complementary nitrogenous bases on the unwound DNA molecule in the 5’ to 3’ direction. This is done based on complementary base pairing.
- DNA polymerase III catalyses the formation of covalent bonds between 2 nucleotides, adding nucleotides in a 5’ to 3’ direction
- As the template strands are antiparallel but the replication fork opens up in one direction only, each new strand needs to proceed in opposite directions
- The leading strand is made continuously, towards the replication fork. The lagging strand is made discontinuously in short Okazaki fragments, away from the replication fork.
- After all complementary bases have been added by DNA polymerase III, DNA polymerase I removes the RNA primers and replace them with DNA nucleotides.
- In the lagging strand, DNA ligase seals any gaps between the nucleotides by catalysing covalent bonds.
Polymerase Chain Reaction (PCR) use and process
- very specific, amplifies a targeted section of the DNA
- only needs a small quantity of DNA
Components needed:
1. Primers - provide a starting point For DNA synthesis.
2. Taq polymerase - a polymerase from bacteria that lives in hot springs, can withstand high temperatures
3. Free nucleotides - for synthesis of the new strand
Process
1. All components are mixed in a tube and placed in a thermocycler which controls the temperature of the reaction.
2. Denaturation - Mixture is heated between 92-98 degC, breaking the hydrogen bonds holding the two strands together
3. Annealing - Mixture is then cooled to between 50-65 degC, allowing the primers to bind with nucleotides on both strands at the 3’ end.
4. Elongation - At 70-80degC, Taq polymerase catalyses the building of new DNA strands by extending the primers
5. Cycle is repeated over and over again until enough target DNA has been produced
Gel electrophoresis use and process
- used to separate fragments of DNA to identify its origins
- DNA profiling: matching unknown sample to known sample. (crime scenes, paternity tests)
Process
1. Restriction enzymes break the long DNA into smaller fragments. If 2 different sources of DNA are to be compared, the same enzymes are used to produce fragments.
2. The DNA fragments are the placed into small wells in a gel in an electrophoresis chamber
3. The gel and sample are exposed to an electric current that is positive on one end and negative on the other.
4. The biggest and least charged particles do not move through gel easily, stick near the wells. Smallest and charged particles pass through the gel to the other side easily.
5. At the end, the fragments leave a banded pattern of DNA.
Proofreading DNA
- DNA polymerase III can recognise mismatched nucleotides and remove it from the 3’ end of the daughter strand.
- After removal of a nucleotide mismatch, it reverses direction and resumes synthesising the new strand.
- DNA polymerase I can also proofread DNA.
