Lecture 1: Cloning Flashcards
What were the issues with old methods of genetics and protein purification?
- Only works for some organisms
- Takes months or years
- Need an obvious phenotype
- Lots of source material
- Years in the cold room
- Human growth hormone
- Factor VIII from blood (HIV)
- Insulin from pigs
What is the overview of the basic cloning reaction?
The basic cloning reaction is a method used to make multiple copies of a gene of interest. There are different ways of cloning
• Restriction enzyme digestion and ligation: most common.
• Topoisomerase-mediated: TOPO cloning uses topoisomerases to hold vectors open and promote ligation.
• Recombination-mediated: Also known as gateway. Uses homologous recombination.
• DNA repair-mediated: Also known as Gibson. Assembly of multiple fragments used in synthetic biology. There is an overlap in the fragments to be joined. An exonuclease chews back the 5’ ends, allowing the two DNA molecules to bind. DNA pol and ligase close the gaps.
• PCR mediated: Fusion. Amplifies the vector and GOI. Adds them together through blunt end ligation.
What are restriction enzymes?
Restriction enzymes are enzymes which cut DNA.
• They were first discovered as part of the bacterial immune system.
• They cut at specific sequences.
• Cleave DNA in a magnesium dependent manner.
• Type II Res are used as they cut at palindromic sequences and cut at or very near the recognition sites on both sides. They are also very precise.
• The length of the target sequence will affect the frequency of cutting. For example HaeIII is a 4 cutter which will cut once every 256 bases assuming random base distribution.
• Some REs have compatible sticky ends. They are isoschizomers. E.g. Sau3a and BamH1.
• Transmethylation protects a restriction site from cleavage.
Why are restriction mapping and RFLPs useful?
Restriction mapping is a technique where the restriction sites of different organisms are mapped. RFLPs (restriction fragment length polymorphism).
• Restriction mapping can show how related different species are by comparing different sites and their positions within the respective genomes.
• RFLPs reflect DNA variations between individuals. If someone has a gene duplication, then it will show in the Southern blot.
• It can also be used for paternity tests due to the inheritance of them.
• Some variation is due to variable number tandem repeats (VNTRs) which have short nucleotide sequences organised as tandem repeats
What is ligation?
Ligation is the process of joining two DNA strands together.
1) AMP is added to an amino group in the enzyme using ATP.
2) AMP is added to the 5’ phosphate. Enzyme is kicked out.
3) The phosphate joins to the 3’OH to join the DNA nick. AMP is kicked out.
An obvious issue with digestion and ligation is that the vector may self-ligate. This is avoided by using a DNA phosphatase which takes off the phosphate from the vector. The insert DNA still has phosphates so it will still work.
Blunt end ligation does not involve sticky ends. It does not require matching sticky ends, but it is about 100 times slower. It therefore requires a higher concentration of ligase.
• Blunt ends can be generated by filling in the overhang. Klenow fragments, NTPs and magnesium can accomplish this.
• Can also be done by removing the overhang.
Why do we use in silico restriction enzyme cloning?
Computer programs are very important for cutting restriction enzyme DNA of interest.
• Snapgene is a very useful program. Gives complete in silico cloning including prediction of restriction digests etc.
• APE (a plasmid editor) is useful for deriving and testing cloning strategies.
