Methods of Genetic Engineering and Analysis
- Created by: Nimnomrata.XOXO
- Created on: 18-02-18 15:34
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- Methods of Genetic Engineering and Analysis
- PCR (Polymerase Chain Reaction)
- The automated process of the amplification of specific DNA regions as well as analysis
- 1. Denaturation
- DNA sample is placed in the cycler and is heated at 94 degrees celsius to form 2 template DNA strands
- The breakdown of the hydrogen bonds between base pairs to form single-stranded DNA
- 2. Annealing
- Temperature decreased to 50-65 degrees celsius so hydrogen bonds can form between bases and primers
- Primers added in high conc, so they can anneal to exposed bases
- Primers are necessary to identify sits where synthesis will take place
- Primers attach to start of DNA strand and then DNA polymerase continues to add nucleotides to rest of chain
- Follows same principle of semi-conservative replication
- Primers may be tagged with fluorescent markers to visualise progress of PCR
- Temperature decreased to 50-65 degrees celsius so hydrogen bonds can form between bases and primers
- 3. Extension/ Elongation
- Temperature is increased to 72 degrees celsius so the DNA polymerase builds up newly synthesised polynucleotides
- Synthesis of polynucleotides are from 5' to 3' direction
- causes exponential growth in DNA population
- Gel Electrophoressis
- Used for the separation of molecules according to their charge/size
- The DNA molecules run through a gel with a buffer solution once an electrical current is provided
- The gel is made of either agarose/ polyacrylamide
- As DNA has the same charge per mass, it is separated according to DNA fragment size
- Due to the charged phosphate group on sugar-phosphate backbone
- negatively charged molecules move towards anode
- Proteins initially denatured and then SDS added so they are separated according to size
- Smaller size, further distance
- Higher charge, further distance
- DNA molecules move through the small pores formed between the hydrogen bonding of the gel molecules
- Pyro-Sequencing
- Process of sequencing DNA, to produce a single-stranded DNA strand
- Complementary to the DNA being sequenced
- 1. Restriction enzymes used to cut sequencing strands
- Fragments formed are 300-800 base pairs long
- 2. Lengths degraded into single stranded DNA (ssDNA)
- Template DNA that are imobilised
- 3. Sequencing primer added
- Incubated with enzymes
- DNA Polymerase
- Luciferase
- Apryrase
- Luciferin
- ATP Sulfurase
- Incubated with enzymes
- 4. One activated nucleotide is incorporated into complementary DNA strand
- Process of sequencing DNA, to produce a single-stranded DNA strand
- Replica Plating
- 1. Pad of sterile cloth pressed on the surface of agar gel on Master Plate
- sterile cloth may also be filter paper
- Some colonies will stick to the sterile cloth
- 2. Cloth transferred on to 2nd petri dish with 2nd marker to see which colonies survive
- Used to identify recombinant plasmids
- If foreign DNA disrupted marker gene, then proper gene product will not be made
- 1. Pad of sterile cloth pressed on the surface of agar gel on Master Plate
- Recombinant Plasmid
- 1. Plasmid cut through use of restriction enzymes
- Each restriction enzyme have their own restriction site
- endonuclease
- Used to identify recombinant plasmids
- A Plasmid that's genome is from two different sources
- 3. DNA fragments form hydrogen bonds with sticky ends
- Complementary Base pair rules
- 4. DNA Ligase bonds the DNA together
- 1. Plasmid cut through use of restriction enzymes
- PCR (Polymerase Chain Reaction)
- 2. Restriction enzyme causes the production of sticky ends
- Sticky ends are exposed bases that are 3-4 bases long
- Recombinant Plasmid
- 1. Plasmid cut through use of restriction enzymes
- Each restriction enzyme have their own restriction site
- endonuclease
- A Plasmid that's genome is from two different sources
- 3. DNA fragments form hydrogen bonds with sticky ends
- Complementary Base pair rules
- 4. DNA Ligase bonds the DNA together
- 1. Plasmid cut through use of restriction enzymes
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