Lecture notes
DNA technology
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- University Of Southampton (UOS)
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DNA TechnologyGene Cloning
Brief history on genetics
1. 1900s Mendelian theory of hereditary
2. 1950-60s Genetic code is cracked
a. DNA is genetic material
b. Transcription and Translation
3. 1970s- Recombinant DNA
Genome- Length of DNA in 1 haploid set of chromosomes- entire gene set
Revolution in Modern Biology
Early 1970s- new genetic research methods
o revolutionised many areas in science
Genetic engineering/ Recombinant DNA technology
Core process- Gene cloning
o Particular gene is copied
What is gene cloning?
1. Gene is Isolated- DNA
2. Inserted into Vector- Recombinant DNA
3. Transported into Host cell
o Typically easy-to-grow, benign, laboratory strain of E.coli
Bacteria
4. Multiplication of recombinant DNA
5. Division of host cell colony/ clones
Plasmid – Based Gene Cloning
Plasmid – carrier of ‘extra’ genetic information not contained within chromosomal DNA and can range in size
from 1 to 100 kilobases
1 kilobase= 1000 nucleotide bases
All plasmids contain origin of replication- they can replicate independently of their host genome
o Plasmid DNA must rely on replication, transcription, and translation machinery of host organism to
replicate/express proteins
Relationship between bacteria and plasmids- mutually beneficial- genes carried on plasmid often confer
survival advantage to organism
Simple cloning vectors
Generate large amounts of DNA
Derived from small circular DNAs- PLASMIDS- found in Bacteria
o Replicate independently of host cell
Why is gene cloning important?
1) Generate large amounts of DNA
a. Large regions of DNA can be made in bulk
b. Pure sample of gene
, c. New combinations of DNA constructed and made
2) Generate large amounts of PROTEIN from gene of interest
3) Gene sequencing & expression
a. Molecular Biology
i. Gene function & regulation in biological process
ii. Tools for knock down & mutagenesis experiment
b. Understanding causes of diseases
e.g. Cystic Fibrosis- inheritance of detective CFTR (Cystic Fibrosis Transmembrane conductor
Regulation) gene
4) Detecting mutations that cause genetic diseases and determining susceptibility to disease
a. Neonatal/ Prenatal Screening- Carried out of occurrence of common genetic disorders- e.g. Cystic
Fibrosis
b. Presymptomatic Testing- for late onset genetic diseases
i. Such as Familial colon Cancer
Huntington’s Disease
Inherited breast and ovarian cancer
5) Develop new ways to treat disease- Production of medically important proteins
a. e.g. Diabetes and Insulin
i. Diabetes- caused by production of insufficient amount of insulin
ii. Previously treated with insulin made from pancreas of cows and pigs
6) Cure inherited diseases using gene therapy
a. E.G. Cystic Fibrosis
Isolating Specifi c Genes
PCR (Polymerase Chain Reaction)
PCR- method on in vitro DNA replication- generate multiple copies of
specific target DNA segment
o Replication reaction is repeated through multiple cycles- exponentially amplify DNA template
o Each cycle of PCR amplification duplicates original DNA template- number of DNA molecules
increases in each cycle
by 2n- n is cycle number
Amplification for 20 cycles result in 220= theoretical max of 1,048,576- identical DNA
molecules starting from single template
o Time required for each cycle is only few minutes – amplification can be accomplished in matter of
hours
Requires:
o ds DNA template
o Pair of oligonucleotide primers
o DNA polymerase
o Nucleotides
Step 1- Denature template DNA to ssDNA with heat at 95oC
Step 2- Lower temperature to allow primers to ANNEAL
o Usually 55-65oC- depending on length and GC content of each
primer
o Annealing temperature- based on Primer melting
temperature (Tm) – can be increased/ decreased within small range to affect stringency of annealing
process
Step 3- DNA polymerase extends primer- produce sequence complementary to each template strand
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