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
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller lisaribau. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for £7.99. You're not tied to anything after your purchase.
