Recombinant and synthetic DNA techniques Flashcards
What is a clone?
A DNA molecule that is genetically identical to the DNA molecule from which it was derived.
What are the uses of a cloned piece of DNA?
Nucleotide sequence can be determined by sequencing, the amino acid sequence can also be determined and you can use this to determine function.
The clone can be used as a probe to determine where and when the cloned gene is expressed.
Gene function can be determined by over-expression or mutation of the cloned gene = functional analysis.
The gene product can be purified for other applications.
The gene can be artificially expressed in other organisms (transgenesis).
What are the main components of the gene cloning toolkit?
Source of gDNA Polymerase chain reaction (PCR) Restriction endonucleases DNA ligase Vector Host
Why do you have to use cDNA to express a eukaryotic gene in a prokaryote?
Prokaryotic genes don’t have introns so they lack the machinery required to remove introns from gDNA.
Which type of restriction endonucleases are most commonly used for gene cloning?
Type II restriction endonucleases.
What is a vector?
A piece of DNA into which the cloned DNA is inserted.
What are the two ways of thinking about gene cloning?
- As a method of identifying a gene in the first place.
2. As a way of manipulating an identified gene.
What are the 3 main methods of gene identification?
- Mapping
- Homology
- Functional complementation of mutants
What is the principle of functional complementation of mutants?
For example to identify a gene responsible for arginine synthesis, arg- mutants are transformed with randomly cut fragments of WT gDNA to find a fragment allowing growth on medium lacking arginine.
For this to work, the whole gene sequence must be in tact after digestion.
1. Chop up DNA into fragments.
2. Ligate fragments into vector.
3. Tranform plasmids into arg- mutants.
4. Select for transformants containing plasmids.
5. Screen for transformants that can grow on medium lacking arginine.
6. Isolate plasmid from colony
7. Sequence DNA fragment inserted into plasmid.
*The fragment capable of rescuing arg- mutants may be quite big so you can perform further digests until identifying a single gene that can restore arginine synthesis.
What is a gDNA library?
A gDNA library is a collection of gDNA fragments ligated into a vector.
What are the 2 ways of digesting gDNA to generate a gDNA library?
Complete digest.
Partial digest.
Which kind of digest is best when the sequence of the gene is unknown?
A partial digest is performed so that the entire gene sequence can be pieced together with overlapping fragments.
How is a cDNA library constructed?
Extract total RNA from a tissue known to express the gene being cloned.
Purify mRNA from the total RNA using oligo-dT cellulose chromatography.
Using reverse transcriptase, convert the mRNA into double-stranded mRNA-cDNA hybrids (this is sufficient for a PCR template) using oligo-dT primer.
Replace the mRNA strand of the cDNA-mRNA hybrid with DNA generating double-stranded cDNA using RNAse H and DNA polymerase.
Add sticky ends (adaptors) to the dscDNA via blunt end ligation.
Ligate the cDNA into a suitable vector to generate the cDNA library.
What is the basis of gene cloning by homology?
We can screen a gDNA or cDNA library based on sequence homology using DNA probes that hybridise to clones of the target genes.
Why is gene cloning by homology useful?
- To identify full-length clone sequences if only the partial sequence is known.
- To identify a closely-related gene in another species.
- To identify the gene sequence from partial protein sequence using oligonucleotide probes.
How do you screen a gene library using a DNA probe?
Beginning with colonies on a plate, each colony contains a plasmid with a particular fragment of DNA.
Transfer colonies to a nitrocellulose membrane and some colonies will stick to the membrane.
Lyse the cells to denature the DNA to give ssDNA.
Incubate the radiolabelled probe with the nylon membrane cotaining all the clones.
The probe will hybridise to clones of target gene.
Expose to an X-ray film and align with the original plate to identify the clone, then sequence for conformation.
Where does the probe DNA come from to screen a gene library based on homology?
May be a fragment of the target gene or protein that may have been obtained by PCR.
It could be a fragment of the same gene from another closely related species.
It could be a very short piece of DNA that corresponds to a fragmet of sequence from the protein encoded by the target gene.
Once we have a full-length clone, what can it be used for?
- Can analyse the cloned gene sequence… for homology with other genes, or to determine the protein sequences and predict structure, or to identify conserved domain.
- Can analyse gene expression profile to determine when and where the gene is expressed by analysing mRNA levels (in situ hybridisation) or fuse it to a fluorescent protein and visualise by microscopy.
- Can analyse gene function e.g. generate mutations and examine the phenotype, over-express the gene, ectopically express the gene.
- Can produce recombinant protein e.g. to perform biochemical characterisation or raise antibodies for detection of the gene product.
- Can make transgenic organisms, express the gene in plants, animals or microbes etc.
What is the benefit of using 2 restriction enzymes when inserting clone into vector?
Directionality.
If the full sequence of a gene is known, is there any need for gDNA or cDNA library?
No, you can use the gDNA/cDNA directly as a template for PCR.
What is the basic method of extracting gDNA?
Any tissue can be used (all tissues have same gDNA).
Homogenise gDNA in lysis buffer.
Add phenol and shake vigorously.
Centrifuge to separate the aqueous and phenol phase.
The phenol phase contains denatured proteins etc.
The aqueous phase contains DNA and partly degraded RNA.
Pipette aqueous phase into a new tube and discard the phenol phase.
Add sodium acetate and ethanol.
Centrifuge and discard supernatant.
This generates a pellet of DNA and RNA.
Dissolve the pellet in water and treat with RNAse to remove RNA and get pure DNA.
What are the uses of gel electrophoresis in gene cloning?
Confirm the presence of DNA/RNA in a sample.
Separate different sized molecules of DNA or RNA.
Determine the size of DNA or RNA molecules.
What is the difference between an agarose gel or polyacrylamide gel?
Agarose gels are used to separate large DNA fragments of 100’s/1000’s of bp whereas polyacrylamide gels are used to separate smaller DNA fragments/proteins when the difference in size is only perhaps 1 nuc in length.
Polyacrylamide gels have much smaller holes.
What is the relationship between the rate of migration and size of a DNA molecule?
The rate of migration of linear molecules is inversely proportional to the log10 of their size.
How does DNA conformation affect the rate of migration?
Supercoiled DNA migrates faster than linear DNA or circular DNA of the same size.
Circular DNA migrates the slowest.
What is the basic method of extracting total RNA?
Take a tissue sample from a tissue known to express the gene of interest.
Homogenise in acid phenol/guanidium thiocyanate solution.
Add chloroform and centrifuge to separate aqueous and phenol phases.
The aqueous phase contains RNA, the phenol phase contains denatured protein and DNA.
Add isopropanol to aqueous phase and centrifuge to give the RNA pellet.
Wash RNA pellet with 70% ethanol and dry pellet.
Dissolve the RNA in water and treat with DNAse I to remove any contaminating DNA.
What is the approximate composition of total RNA?
80% rRNA
15% tRNA
2-5% mRNA
What feature of mRNA is used to capture mRNA from total RNA?
The polyA tail, a sequence of approx 200 A residues at their 3’ end.
