L1 Recombinant tech 1 Flashcards
(13 cards)
Recombinant DNA Adv - 3
Adv:
1. Efficient, cheap, & safe production, e.g. insulin, factor VIII
2. Makes rare proteins with therapeutic potential in quantities for pharmaceutical value, e.g. interferon
3. Production of vaccines, e.g. hepatitis B virus, SARS-CoV2, HPV
Recombinant DNA in nature & technology - 5
In natural processes:
1. DNA repair
2. Acquire new functions such as multi-drug resistance
Recombinant DNA technology:
3. Analyse function of genes and their products
4. Expression/regulation studies
5 Production of industrial & pharmaceutical products
Transcription - 5
- RNA polymerase moves along the DNA unwinding the strand, starting at 5.
- Hydrogen bonds between base pairs break which allows the unzipping of the double helix.
- As RNA polymerase breaks the bonds, it synthesises aprimarytranscript of mRNA using RNA nucleotides. These form hydrogen bonds with the exposed DNA strand by complementary base pairing.
- The primary transcript of mRNA is processed to produce amaturetranscript of mRNA.
- The mature mRNA transcript is now ready to leave the nucleus and travel to the ribosome.
Translation - 7
- mRNA molecule travels through cytoplasm & attaches to the ribosome.
- tRNA molecules transport specific amino acids to the ribosome.
- Each mRNA codon codes for a specific amino acid.
- The anti-codons & codons match up & form complementary base pairs.
- Peptide bonds form between adjacent amino acids to form the polypeptide.
- Used tRNA molecules exit the ribosome & collect another specific amino acid.
- The last codon of an mRNA molecule is a stop codon which signals the end of translation.
Natural transfer of DNA - 3
Transformation: uptake of free DNA (competence), most used in labs
Conjugation: transfer of DNA through cell-cell contact
Transduction: transfer of DNA mediated by a virus
N.B. for animal cells, equivalent of transformation is called transfection
Mobile genetic elements
Mobile genetic elements: bits of DNA which are efficient in transferring from one to another e.g. plasmids, transposons, prophages
Plasmids - 3
Naturally occurring plasmids are not essential, but often encode genes that are helpful
These plasmids may be involved in e.g.:
- resistance to antibiotics or toxic metals
- metabolic functions (e.g. growth on lactose, sucrose)
- production of virulence factors (e.g. toxins such as haemolysin)
Molecular cloning - 2
- Obtain a defined sequence of DNA & produce multiple copies in vivo
- The DNA sequence can be a gene, but may also contain non-coding elements e.g. promoter
Three basic steps in molecular cloning
- Isolation of source DNA
- Inserting source DNA into a cloning vector
- Introduction of cloned DNA into a host organism
Molecular cloning - 7
- DNA fragment is isolated, then using enzymes is inserted into plasmid vector
- Mix recombinant cell with E.coli (easy to transform) in the presence of CaCl2, providing a heat pulse
- CaCl2 is used as both membranes are negatively charged
- Heat pulse generates holes, pushing E.coli to be competent for DNA uptake
- E.coli takes up plasmid (which contains a ampicillin resistance gene)
- The cells are placed in presence of ampicillin, only transformed cells can grow due to resistance
- The plasmid replicates in the plate, ensuring each cell which grows is transformed
Obtaining DNA for cloning: A or B
A) Sequence of DNA known:
Polymerase Chain Reaction (PCR) is the most common technique
B) Sequence of DNA unknown:
May require the creation of a DNA library, followed by “fishing” for the gene of interest
PCR - 4
Method to amplify section of DNA
Three steps, which are repeated 25-35 times:
1. Denaturation of DNA strands (~30 sec at 94 oC)
2. Annealing with primers (~30 sec 55-65 oC)
3. Elongation with thermostable DNA polymerase (~1 min per kb at 72 oC)
4. Repeat several times
PCR steps - 6
- Identify target region (e.g. a specific gene)
- Design primers that are complementary to the red and green regions
- Heat up to 94 degrees for 30s, DNA is denatured
- Heat up based on primers so the primers can interact with complimentary sequences
- Thermostable Taq polymerase starts at 3 end and moves to 5, binding nucleotides to make new DNA
- The process is repeated roughly 30 times
