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Topic 1: Isolating and Amplifying specific DNA fragments (In vivo vs. In vitro)
Key words to mention to the class so they don’t get lost during presentation:
Amplification: replication done to DNA segment we’re looking at
o Can be performed with bacterial cells or in test tube (in vitro)
Donor DNA: the sample DNA we’re using, sometimes is the entire genome
In Vivo:
Parts of the Donor DNA are added into the plasmid or bacterial virus that will take over and
amplify the gene we’re looking at. This will be called Vectors.
1. The Donor DNA are cut up using enzymes called restriction endonucleases as their
molecular scissors
2. The scissors cut the DNA into smaller fragments
3. Each little fragment is added into vector chromosomes already cut to form recombinant
DNA molecules
4. These recombinant DNA are transferred into bacterial cells - which is taken by each cell
In Vitro:
Is performed by PCR (Polymerase Chain Reaction):
It recognises the location of the gene by complementation on the short primes
Amplifies it by cycles to create many of them
Both of the techniques require specification of a protein to notice the sequence of interest and
also the formation of a ds molecule from a starting ssRNA/ssDNA.
Define Sources of Donor DNA (what we’re using the amplify and isolate DNA)
Genomic DNA: This is the entire genome. gDNA obtained directly from chromosomes of
organism of interest. Before any cloning can be done, it must be cut up.
cDNA (complementary DNA): double stranded version of an mRNA molecule, is DNA
copied by mRNA. Doesn’t need to be cut in order to be cloned. cDNA is created from
mRNA with the use of reverse transcriptase, an enzyme that was isolated from
retroviruses. The reason why it’s more useful to use cDNA in eukaryotes is because the
introns have been spliced out in the genomic sequence.
Chemically synthesized DNA: a specific recombinant DNA that cannot be isolated. If
the DNA sequence is known, then the gene can be chemically synthesized
DNA is cut in small fragments of interest (restriction fragment) by using restriction enzymes that
have a sticky end.
Hybridization
DNA palindrome (the strands are the same just in different directions) is cut by the restriction
enzymes mostly stagger (offset). In EcoRI there are 6 nucleotides and when the palindrome is
cut each strand has 5 nucleotides and one sticky ends. Under specific conditions, Donor DNA
can be inserted by complementing the sticky end and by using DNA ligase to seal them.
PCR In Vitro:
Multiple copies of short primers binds to different ends of genes or regions to be
amplified. Two primers bind to opposite DNA strands. Polymerases add bases to the
primers and polymerization proceeds back and forth.
Cycles of denaturation ( high and low), annealing and synthesis are repeated in the
amplification process.
DNA polymerase Taq polymerase, comes from the bacterium Thermus aquaticus, is
extremely heat resistant.
Kary Mullins made PCR possible and awarded Nobel Prize in Chemistry 1993
Cloning DNA fragments with blunt ends: “ connects harder because ddDNA”
I.e. cDNA and DNA fragments have blunt ends that come from PCR.
Not effective: blunt end fragments joined to the vector with only ligase.
Effective ways:
1. Create sticky ends of PCR products using specific PCR primers that contain
endonuclease recognition sequences at 5’ ends. The last PCR product is digested with
the restriction enzyme and produces a fragment to be inserted into a vector.
2. Add sticky ends to double stranded oligonucleotides called linkers or adapters that
contain a restriction site. Linkers are joined by ligase to cDNA strands. Then to get sticky
ends for cloning into a plasmid vector, DNA is incubated with the restriction enzyme.
Finding DNA
After the cDNA are created we need to find our recombinant DNA of interest
We can localize it by using probes, which are small derivative of relative cDNA (for
complementation) or protein that is produced by the gene of interest (from protein to mRNA to
gene of interest)
Probes can recognise:
A nucleic acid
Specific protein
→ Autoradiogram
Everything consists in complementation so the molecules are ss.
The vectors are on a petri dish and a membrane containing probes (usually radioactive isotopes
or fluorescent dyes) for complementation is above the vectors. The membrane is removed and
put on a X-ray film to notice the location. So we go back to the petri dish and now we know
which spots contain the gene of interest to make a colony of it in order to amplify it.
Finding Protein
We can find the gene by knowing the protein that it produces. All our clone vectors are in the
petri dish and they are going to produce “fusion” proteins. We lay a membrane above it and we
transfer the membrane containing the proteins in a petri dish with antibodies to recognise the
gene that created the protein. We use “secondary antibodies” to localize the primary antibody by
using X-ray film.
In order to determine the nucleotides and its biochemistry in the gene of interest we perform the
technique of Sanger sequencing (dideoxy sequencing). It consist in the ability to stop the DNA
synthesis by the present of a modified nucleotide dideoxynucleotide (~ddNTP).
In a primer DNA we add DNA pol, 4 dNTP (dATP, dTTP, dCTP and dGTP) and 1/chase ddNTP
(ddATP/ddTTP/ddCTP/ddGTP). So depending on the ddNTP we are using we are going to stop
the DNA synthesis at the dNTP, eg: ddCTP will stop when it added instead of dCTP. We are
going to have 4 reading (1/N) that is going to reveal us the location in the DNA sequence. If we
have an automatic sequencer we proceed the DNA sequence once and simultaneously the
location of 4N will appear.