MCG - Manipulation of DNA, use in Therapy and Diagnosis Flashcards
List some benefits to having the ability to isolate and manipulate DNA.
- isolate and modify genes to understand their function
- go from mutant phenotype (disease) to gene
sequence - manufacture proteins (insulin, factor VIII etc)
- generate vaccines
- create transgenic plants or organisms
- diagnose genetic diseases
- develop gene therapy
- allow move to personalised medicine by identifying genetic changes leading to disease in an individual
What is Recombinant DNA technology?
Recombinant DNA technology (or Gene Cloning) allows the isolation and manipulation of DNA.
This is made possible by using methods to isolate sections of DNA and then create multiple copies of these sections of DNA.
It is known as Gene Cloning because copies (or clones)
are made of unique pieces of DNA.
Describe restriction enzymes.
Restriction enzymes function as ‘molecular scissors’ to cut DNA.
They do this because restriction enzymes recognize specific nucleotide sequences in the DNA and cuts both strands of the sugar-phosphate backbone.
It was first identified in bacteria where they protect against viral infection by cutting (restricting) viral DNA.
Describe what happens at the restriction enzyme sites.
Different restriction enzymes recognize different nucleotide sequences.
The sequence forms a palindrome (sequence reads the same on both strands in 5’-3’ direction).
The restriction enzymes cut in a characteristic cleavage to leave cohesive (sticky) or blunt ends.
How is DNA visualised in agarose gel electrophoresis?
DNA is visualised in gel by adding ethidium bromide which binds DNA and fluoresces when exposed to UV light.
What is recombinant DNA, and why do we need it?
They are large amounts of individual DNA fragments that are needed to allow further study, manipulation or to develop therapeutics.
They are used in bacteria or yeast to replicate and amplify individual
DNA fragments.
We need mechanism to introduce DNA into the host organism.
Plasmids (circular DNA molecules) are used as ‘cloning vectors’ to carry DNA fragments into host.
Describe how we can clone DNA molecules.
A DNA molecule or gene can be cloned by inserted it into a plasmid.
Plasmids contain an origin of replication that allows replication independently of the bacterial chromosome.
This allows for high copy numbers of plasmids, around 100-200, to be maintained in each bacteria.
Plasmids also contain antibiotic (AB) resistance genes that allows forvthe selective growth of bacteria that contain plasmids.
Describe the process of creating a recombinant DNA molecule.
To clone a DNA fragment, the fragment and plasmid are cut with the same restriction enzyme.
This means that both plasmid and fragment will have same complementary cohesive ends.
The cut DNA fragments and cut plasmid are mixed together.
DNA fragments will anneal to cohesive ends in plasmids.
The DNA ligase will ligate the DNA fragment and plasmid together to form recombinant DNA molecule.
What is a genomic library?
A genomic library is a collection of plasmids each with a different DNA fragment.
The collection is sufficiently large so that each fragment from the genome is represented.
List some uses of cloned DNA.
- to clone and piece together genomic DNA to allow the mapping and
sequencing of genes within the genome. - to identify changes in the genome that are associated with particular
phenotypes, pathologies or diseases. - to characterise how the genome is organised, location of repetitive DNA etc
- to genetically engineer organisms by transferring genes between organisms
- to allow for gene therapy or gene replacement treatment
- to express large amounts of protein from isolated genes to allow study
or production of new therapeutics (biologics). - to identify genes that are expressed in different cell types, i.e different cells in the same organism or genes expressed differentially in healthy and diseased organisms
What is cDNA, and how is it used?
To express eukaryotic proteins or identify genes expressed in a particular cell type, we need to make DNA copies of the transcribed mRNA.
mRNA is produced when genes are transcribed, and it does not contain introns.
However, we cannot clone RNA, only DNA.
Complementary DNA (cDNA) is a DNA copy of a mRNA, produced using an enzyme called reverse transcriptase.
cDNA libraries contain complementary DNA copies of the mRNAs present in a cell population and represent the genes being expressed in the population from where the mRNA is harvested.
cDNAs are used as the introns are removed and the clone size is smaller.
Describe DNA sequencing and its uses.
Full characterisation of genes within the genome can be obtained by sequencing our DNA.
This provides the sequence of bases which can be used to predict the sequence of amino acids contained in the protein that the gene encodes.
This allows us to determine some properties of the gene product and identify changes that cause disease.
DNA sequences from different individuals can also allow us to discover genetic differences, which is the first step to personalized medicine.
Describe the method of DNA sequencing.
The DNA sequencing method devised by Sanger is the most common method (Nobel Prize 1980).
It uses DNA polymerase to copy single stranded DNA. It makes use of ‘dideoxy’ nucleotides, which interrupt the ability of DNA polymerase to copy DNA.
DNA polymerase cannot continue to extend the chain of
nucleotides after the incorporation of a dideoxynucleotide.
This method known as the dideoxynucleotide chain-termination sequencing.
This method can be automated and allows rapid sequencing of large amounts of DNA.
What is the difference between deoxynucleotides and dideoxynucleotides?
Deoxynucleotides have an –OH, whilst do not have a –OH on the 3’ carbon.
DNA polymerase cannot incorporate any further nucleotides after incorporation of a ddNTP as there is no 3’ OH to form the phosphodiester bond.
What is automated DNA sequencing?
Automated DNA sequencers use a single reaction in which all four ddNTPs are included together with dNTPs.
Each ddNTP is labeled with a unique fluorescent marker. We can identify which ddNTP was added at the end of each fragment by identifying its fluorescent colour.
The DNA sequence fragments are separated on a capillary gel.
The fluorescent label on each ddNTP has a different wavelength. As the fragments move down the gel it passes a laser - smaller fragments move faster.
The laser light excites the fluorescent tag on each fragment as it passes. The wavelength of the fluorescence is read as the fragment passes, and the information is recorded.
The fluorescence pattern produced shows the sequence of the DNA.
