Lecture notes from Imperial College London, Medical Biosciences BSc, 2nd year, genetics and genomics (GEN) module.
Focus on genomic DNA. How do we know it is the DNA that genes are made of? In fact, for a long time it was thought that genes must be made of protein. We'll describe how this idea w...
Defining the Genome II - DNA
- for a long time, we thought that genes were made of protein
DNA is the genetic material
- Fred Griffiths discovers in 1928 that DNA is responsible for heredity
=> 2 ≠ stains of bacterium causing pneumonia: Strain S (pathogenic) & Strain R (non-pathogenic)
=> D
(DNA)
=> R strain was transformed into S strain by something in mucus (transformation principle)
=> S-type was heritable => transforming principle must be genetic material => DNA
=> DNA can be used to genetically modify an organism
Methods to analyse DNA
- Sanger sequencing: 1970 (Fred Sanger)
=> determine sequence of a DNA fragment (max 1000 nucleotides) prepared by cloning/ PCR
=> uses 1) target DNA 2) oligonucleotide primer 3) DNA polymerase 4) dNTPs + ddNTPs
=> DNA polymerase extends primer (w/ all dNTPs) until a ddNTP (lack 3’-OH) is incorporated -
fragments separated w/ electrophoresis (≠ lengths) - coloured DNA (if fluorophore tag)
Rq: nucleic acid = base +
phosphate + sugar
fluorophore tags
=> I
use tube
if no
tag => 4 tubes with 4 F ddNTPs
- Restriction enzymes: 1970
=> endonuclease enzymes that cut DNA at phosphodiester bonds (unable to cut methylated DNA)
=> leave free 3’-OH and 5’-phosphate groups
, => come from bacteria (response to viral invasions)
=> recognise 4-8 bp target sequences + can cut smaller fragment by targeting restriction sites
=> restriction enzyme EcoRV cuts DNA
at GATATC
=> no overhang: “blunt end”
=> restriction enzyme Hindlll cuts DNA at
AAGCTT (palindromic)
=> singe-stranded overhangs: “sticky ends”
- recombinant DNA cloning: 1972
=> DNA inserted into a plasmid (circle of DNA in bacteria, independent from chr)/ vector
=> DNA + plasmid digested with the same restriction enzyme
=> annealing + ligation of sticky ends/ ligation of blunt ends
=> DNA ligase join DNA and plasmid together
=> introduced to bacteria: clones w/ same recombinant plasmid
// reproductive cloning
= nuclear transfer into
enucleated zygote
=> used to sequence 1st human genome: DNA was fragmented, cloned into plasmid + inserted
into bacterial cells - plasmids were replicated for Sanger Sequencing
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