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DNA technology (PCR (Step 1: Separating the strands
Temperature: 94
30…
DNA technology
PCR
Step 1: Separating the strands
Temperature: 94
30 seconds
Denatures DNA by breaking hydrogen bonds that holds strands together
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Step 3: Synthesis of DNA
Temperature: 72 (optimum temperature for DNA polymerase)
1 minute
DNA polymerase adds bases to the primer making a complementary strand and creating an identical double helix DNA to the first.
Taq polymerase is used (from thermophilic bacteria from hot spring)
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Simple analysis
- A number of the same BAC sections are takens and treated with different restriction enzymes
This gives a number of overlapping fragments
- The fragments are separated using electrophoresis which separated them into size order
- Each fragment is sequenced
- Computer programs are used to reassemble the full BAC sequence by analysing the overlaps shown by the fragment sequences.
Satellite DNA
Minisatellites (VNTR's)
20-50 base pairs, repeated 50 to several hundred times
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Genetic engineering
- Identify gene
- cut out using restriction enzymes (creates sticky ends)
- Cut plasmid (vector) with same restriction enzyme
- Insert gene into plasmid (has marker genes) using DNA ligase (gets recombinant DNA)
- Insert plasmid into bacteria by placing them into a calcium ion rich solution and raising the temperature (Transformation)
- Isolate bacteria with plasmid with inserted gene
or
- isolate mRNA (introns cut out)
- use reverse transcriptase
- DNA polymerase
Or
- Create gene
- then same insertion process
Electrophoresis
- DNA segments put into wells of gel
- Buffer
- DNA moves towards positive electrode. Smallest fastest
Hybridisation
- use gene probe
- small piece of single strand DNA with complementary base pairings
- anneals to complementary bass
- have radioactivity or fluorescent dye
DNA profiling
- Extract DNA
- PCR (creates larger sample of DNA)
- hydrolyse (digest) sample using restriction endonucleases (Cutting DNA into fragments)
- gel electrophoresis to separate fragments
- Transfer fragments from gel to nylon membrane using Southern blotting
- Hybridisation (Radioactive/fluorescent DNA probe added to label specific fragments)
- Development (the nylon sheet is placed onto a X-ray film and reveals dark bands where to DNA probes are attached)
Clone library
- the length of digested DNA are place into bacterial artificial chromosomes (BACs)
- Placed into E.Coli and cultured (cloned)
- to create a clone library
Vectors
Plasmid, BACs, Yacs, Viruses (T4 phage) or liposomes (or bacterias for plants)
Virus and liposomes best for human
Uses for sequencing
- comparing genomes to look for common genes in individuals and species and evolutionary relationship
- predict potential amino acid sequences
- modelling gene changes (use common genes found in human/yeast cells)
- compareing genes from pathogens and non-pathogens to get information for new drugs and treatment
- Synthetic biology: deisgn and construction of new biological entites such as enzymes, genetic circuits and cells or the redesign of existing biological systems
- bioinformatics and computational biology used in studying genotypes-phenoypes relationships, epidemiology and evolution
-forensics / paternity/disease risk
- humand genome project
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- Transformation can also be done by Electrofusion for cells
- Tiny electric shocks to the cell membrane will cause the cell and nuclei to fuse
- Easy in plant
- Harder in annimals but good for monoclonal antibodies cells
and
- elctroporation
- small current make bacterial membrane permeable plasmids can move in
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