what’s DNA ligase
enzyme that catalyses the joining of sugar and phosphate groups (phosphodiester) within DNA via condensation reaction from 3’ to 5’
DNA polymerase vs DNA ligase
DNA polymerase adds nucleotides to the template strand (5’ to 3’), and ligase seals the gaps between the nucleotides on lagging strand (3’ to 5’) by joining Okazaki fragments
what are plasmids
small loops of DNA in prokaryotic cells
what is Recombinant DNA
a composite DNA molecule created in vitro by joining foreign DNA with a vector molecule (plasmid)
What’s genetic engineering
when genes are isolated from one organism and inserted into another organism, using vectors.
What are the main steps in genetic engineering
- The required gene is obtained
- A copy of the gene is placed inside a vector
- The vector carries the gene into a recipient cell
- The recipient expresses the novel gene
Step 1 - How is the required gene obtained in genetic engineering
- mRNA obtained from cells where the required gene is being expressed. Reverse transcriptase catalyses formation of a single strand of complementary DNA (cDNA) using the mRNA as a template. The addition of primers and DNA polymerase can make this cDNA into a double-stranded length of DNA, whose base sequence codes for the original protein.
- If scientists know the nucleotide sequence of the gene, then the gene can be synthesised using an automated polynucleotide synthesiser.
- If scientists know the sequence of the gene, they can design PCR primers to amplify the gene from the genomic DNA.
- A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes.
what’s a vector
in gene technology anything that can carry/insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria.
Step 2 - How is the gene placed into a vector
- Plasmids mixed with restriction enzymes that will cut the plasmid at specific recognition sites.
- The cut plasmid has exposed unpaired nucleotide bases, called sticky ends
- If free nucleotide bases, complementary to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal. DNA ligase enzyme catalyses the annealing.
- A gene may be sealed into an attenuated (weakened) virus that could carry it into a host cell.
What are the 5 methods used to insert vector into the recipient cell
- Heat shock treatment
- Electroporation
- Electrofusion
- Transfection
- T1 (recombinant) plasmids
What’s electrofusion
electrical fields help to introduce DNA into cells.
What’s electroporation
a pulse of electricity makes the recipient cell membrane more porous.
what’s heat shock treatment ?
- bac subjected to alternating periods of cold and hot in the presence of calcium chloride,
- their walls and membranes will become more porous and allow in the recombinant vector.
- Ca2+ reduces repulsion between -vely charged parts of DNA and phospholipids
what’s transfection
DNA can be packaged into a bacteriophage (a virus which parasitizes a bacterium by infecting it and reproducing inside it), which can then transfect the host cell.
How are T1 (recombinant) plasmids used to get vector into recipient cell
the plasmids are inserted into the bacterium Agrobacterium tumefaciens, which infects some plants and naturally inserts its genome into the host cell genomes.
How is reverse transcriptase used
- Find a cell that produces the protein you require (e.g β cells of the Islets of Langerhans in the pancreas produce insulin).
- Extract the mRNA from the cells.
- Use reverse transcriptase to make cDNA from the mRNA.
what’s the direct method of introducing gene into recipient
- If plants are not susceptible to A. tumefaciens, then direct methods can be used.
- Small pieces of gold or tungsten are coated with the DNA and shot into the plant cells. This is called a “gene gun”.
What’s genetic engineering also known as?
- recombinant DNA technology
- genetic modification
function of reverse transcriptase found in retroviruses (HIV)
- HIV contains RNA that they can inject into the hosts genome
- reverse transcriptase catalyses formation of cDNA using RNA as template
- useful in genetic engineering
restriction enzymes create ……… and ……….. ends
- sticky (staggered cuts)
- blunt (cuts not staggered)
How is insulin obtained from GM bacteria
- mRNA obtained from B cells
- adding reverse transcriptase enzyme makes a single strand of cDNA and treatment with DNA polymerase makes a double strand - the gene
- addition of free unpaired nucleotides at the ends of DNA produces sticky ends
- with the help of ligase enzyme, the insulin gene can be inserted into plasmids extracted from E.coli bac ( now recombinant DNA)
- Bac mixed with recombinant plasmids and subjected to heat shock in presence of CaCl2, so they’ll take up the plasmids
what are the two antibiotic resistance genes in bacterial plasmids
- resistance to tetra
- resistance to amp
How do you find the plasmids that have taken up the desired gene?
- desired gene is inserted into the plasmid midway through the gene for tetra resistance.
- Some plasmids take up the gene some do not
- all colonies grow in amp plate (as all plasmids have gene for amp resistance)
- Bacteria only grow on the tetracycline plate if they have NOT got the insulin gene.
How is the risk of transgenic bacteria escaping into the wild controlled?
- genes knocked out so they can’t synthesise particular nutrients so will only survive in the lab where they are supplied those nutrients in growth medium
