Manipulation of DNA, use in therapy and diagnosis Flashcards
Ability to isolate and manipulate DNA allows us to
isolate and modify genes to understand their function
go from mutant phenotype to gene expression
manufacture proteins, vaccines (insulin)
diagnose genetic diseases
develop gene therapy
PERSONALISED MEDICINE
Recombinant DNA technology
allows for isolation and manipulation of DNA
Due to gene cloning- isolate sections of DNA then create multiple copies of these sections
i) need a method to isolate pieces of DNA eg restriction enzyme, PCR
ii) need method to copy DNA- inserting DNA into plasmids so they can be replicated
Restriction enzymes
function as molecular scissors to cut the DNA- specific enzymes recognise the specific nucleotide sequences in the DNA Cuts both strands of the sugar phosphate back bone
Restriction enzymes can produce cohesive ends, blunt ends, cut into small fragments
Issues with recombinant DNA
large amount of recombinant DNA fragments needed to allow further study therefore use bacteria/yeast to replicate and amplify individuals DNA fragments
Need mechanism to introduce DNA into host organism
Using plasmids- “cloning vectors” to carry DNA fragments to the host
Cloning DNA Molecules- obtaining recombinant DNA
1) DNA fragment and plasmid are cut with the same restriction enzyme. This ensures that both the plasmid and the DNA fragments will have complimentary cohesive ends
2) Cut DNA fragments and cut plasmids are mixed together
3) DNA fragments anneal to cohesive ends in plasmid- allows replication to occur independently, high number of plasmids to be maintained in each bacteria
4) DNA ligase will ligate the DNA fragments together
RECOMBINENT DNA MOLECULE
Cloning DNA Molecules- Amplification
Individual recombinant plasmids can be taken up by Ecoli bacteria-
each bacteria contains 100-200 copies of the recombinant plasmid
Examples of uses of cloned DNA
Clone and piece together genomic DNA to allows mapping and sequencing of genes within the genome
Identify the changes in the genome that are associated with particular phenotypes, pathologies or diseases
Characteristise how genome is organised, location of repetitive DNA
genetically engineer organisms by transferring genes between organisms
Allow gene therapy
How is cloning with eukaryotes different ?
Difficult to clone because the eukaryotic genes contain introns and so one cannot express the protein using genomic DNA
Instead using mRNA where the introns are removed and convert this back to DNA (Reverse transcriptase)
Different cells express different genes ie not all cells express the same genes. Harvesting mRNA from different cells allows us to discover which genes are expressed by which cells- how this changes in disease
This is why cDNA is used- mRNA does not contain introns
which is why it cannot be cloned. Only DNA can be.
cDNA instead is used which is a DNA copy of mRNA, using reverse transcriptase.
DNA Sequencing
Obtaining a full characterisation of genes within the genome
Provides the sequence of bases- predict the sequence of amino acids contained in the protein. Therefore determines the properties of the gene product and identify the changes that cause disease
Goes back to personalised medicine
Method of DNA Sequencing
DNA polymerase- copy single stranded DNA
The use of “dideoxy nucleotides” which interrupt the ability of DNA polymerase to copy DNA. Therefore DNA polymerase cannot continue to extend the chain of nucleotides after incorporation of dideoxynucleotide
RAPID and Automated -
when DNA is replicated by DNA polymerase in presence of dCTP and small amount of ddCTP some fragments end prematurely. The length of the fragments depend on when ddCTP is incoperated
why are dideoxynucleotides used”
do not have the -OH at the 3’ end
Hence why the DNA polymerase cannot incorporate any further nucleotides after incorporation of a ddNTP
Automated DNA sequencing
Single reaction in which all the 4 ddNTPs are included together with DNTPs
1) ddNTPs labelled with fluroescent marker
2) DNA segments separated on a capillary gel
3) The fluorescent label on each ddNTP has a different. wavelength
4) As the fragments move down the gel, it passes a laser, smaller fragments move faster
5) The wavelength of the label is read as fragment passes
6) The fluorescence pattern produced shows sequence of DNA
