Produce a large number of identical DNA molecules in a bacteria.
Examples
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Human insulin genes
Human growth hormone
Tissue plasminogen activator
Factor VIII (prevent haemophilia)
Hepatitis B virus vaccine (basically surface protein antigens)
Vector DNA cloning
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1. Restriction enzyme celaves double stranded DNA.
2. Ligation of DNA with cleaved plasmid.
3. Recombinant Plasmid is slammed into the cell. By CaCl2 at 4 deg and heatshocked at 42deg for 2 minutes.
Either this or electroporation.
Antibiotic resistance determine if the bacteria survive
The colour determine if the bacteria had the betagalactosidase to break down the lactose, if there was, it means the plasmid did not bind properly
Plasmids multiply as the bacteria grow.
Bacteriophage
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Based on lambda phage.
only a certain size of insert from 15-21kb is able to be cloned.
Properties
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1. Origin of Replication
2. High Copy Number of vector in E.coli cell.
3. Vector is Small
4. Multi-cloning site for restriction enzymes
5. Antibiotic resistance genes
Artificial Chromosomes
Bacterial Artificial Chromosomes
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Similar to pUC19, different origin of replication.
Able to accommodate up to 100,000 inserts.
Yeast Artificial Chromosome
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Clone very large segments of DNA. 50,000 to 1000000 base pairs.
They need to have a bacterial origin of replication and antibiotic resistance gene.
Alkaline Lysis
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1. Cells are lysed by SDS
2. DNA made single stranded by NaOH.
3. DNA neutralized and only plasmid remains.
4. Precipitates removed by centrifugation
5. RNA lysed by NaOH and RNase.
Libraries
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Presence of the DNA sequence is logged in the libraries.
Genomic DNA clone
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Contains every Human DNA sequences
mRNA clones
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Only expressed sequence is derived from mRNA.
mRNA is copied into DNA using reverse transcriptase.
cDNA is the result, and it does not contain introns.
Reverse transcriptase goes from 5 to 3 in synthesis. RNA is removed by alkali and resyntehsis again,
Identification of Recombinant DNA
DNA hybridization
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Identification was done by autoradiography.
A labelled probe was inserted in the colonies. Those who can bind to the colonies such as 32P(radioactive)GATC can bind to the selected product.
Process
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1. Colony hybridisation: Done via Southern Blotting Process.
2. Plate of bacterial colonies are transferred to a membrane forming a replica of bacterial colonies
3. Alkali lysed and DNA is made single stranded
4. Probe inserted.
5. Washing away unhybridized probe. Autoradiography is used to determine desired sequence.
DNA Probe
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Oligonucleotides are synthesized from machine and uses 5' end to do radioactive labelling
Expression of protein product
Process
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Almost the same as the DNA vector
Blotting
Northern Blotting
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Determines and analyses RNA
Process is same as southern blotting.
Isolate RNA from cells
Electrophorisis on Agarose Gel
Blot into Nylon filter
Hybridize with 32P DNA probe, determine size of DNA
Techniques
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DNA sequencing and PCR is one of the the 2 techniques that depends DNA Polymerase.
REcall, DNA adds on from 5' to 3'.
Termination occurs when ddNTP is incorporated. Therefore it will not be extended.