Term 2 Lecutre 6: Recombinant DNA Technologies Flashcards
Sometimes when isolating a gene a genomic DNA library isn’t a good starting point
1) if a genome is large the library will also be very large and therefore many more colonies would need to be screened to find the specific gene
2) some genes are very large because they have many introns and may be difficult to clone into a plasmid (in this case phage lambda or cDNA would work)
3) or if the gene is to be expressed (to produce protein) in bacteria but it is from a eukaryote and contains introns, bacteria cannot remove introns and therefore the protein could not be produced from a genome sequence
^ in this case complementary DNA (cDNA) is the answer. cDNA is synthesised from mRNA and therefore only includes expressed gene sequences (coding regions)
A cDNA population is uniqe
It is unique to the tissue, age and environmental conditions etc. Of the sample from which the mRNA was isolated.
So for example cDNA libraries made from plants of different ages/stages of development would all be different because certain genes are expressed at each stage.
Synthesising cDNA
1) isolate mRNA and use polyA tail to link an oligo(dT) primer
2) this primer allows you to synthesise in 5’ to 3’ direction by reverse transcriptase a copy of the 5’ 3’ ssRNA
3) ribonuclease used to remove RNA from genomic DNA samples
4) in this example some RNA is left to act as primers for DNA polymerase
5) dscDNA molecule is formed
Cloning cDNA
After cDNA synthesis the cloning steps are the same as for a gDNA library. The screening with a labelled probe is also the same.
Key difference: conversion of mRNA to cDNA fragments ready for use whereas gDNA is instead digested to make fragments for use
cDNA fragments have blunt ends (not sticky) so restriction enzymes that cut to produce blunt ends are used to open the vectors (plasmids)
If you are interested in the regulatory elements of a gene such as when/where expressed you need gDNA library
E.g. if you want to isolate the promotor region of a gene to figure out where/when it is expressed by linking that promotor to a reporter gene for example such as GFP to find and follow gene activity
To do this you must isolate from genomic DNA because that would not be present in cDNA.
Same would be the case to check how a gene was spliced as in that case you would need the introns inckuded
So when would you be interested in isolating a cDNA fragment?
If you would like to express the gene (produce a protein) then you would use mRNA/cDNA (cDNA library)
To observe levels of gene expression i.e. mRNA or transcript abundance in:
-different cell types
- different developmental stages
- cells grown under different conditions
-normal Vs disease states
^ you wouldn’t get any of this info from gDNA as gDNA stays the same in all cells
cDNA can also be used as expression libraries - in this case an antibody can be used to screen for a gene product i.e. the expressed protein
Screening an expression cDNA library
mRNA is converted to cDNA and cloned into an expression vector and added to bacteria (E. coli). The transformed colonies are grown on agar plates. The bacteria are lysed and transferred to nitrocellulose, radiolabelled antibody is added that binds specifically to the protein of interest. Dark spots on an autoradiogram confirm presence of desired protein.
Often genes are isolated directly from cDNA by PCR - in this case a cDNA library is not required (a simpler method)
Rather than cloning it into plasmids and then screening it you can use that DNA to isolate the gene by PCR
PCR with gDNA compared to PCR with cDNA
PCR gDNA
Genomic DNA template, gene specific primers added to a tube of dNTPs and PCR buffer and set PCR machine to run to isolate fragments
PCR cDNA
Very similar but at the start you have RNA which cannot be amplified by PCR (only DNA can be) so first the reverse transcriptase reaction is required to convert mRNA to cDNA
So step 1 - RNA from sample+MMLVRT enzyme+poly dT primer mixed with dNTPs and PCR buffer to convert mRNA to cDNA
Step 2: PCR reaction to amplify gene of interest
This can all be done in one machine - the PCR machine regulates its temperature accordingly
^ a powerful technique for gene expression studies
Cloning summary
gDNA:
Isolate DNA to clone → separate strands+add primers→PCR
→gDNA clones
cDNA:
Convert mRNa to cDNA: add first primer, reverse transcriptase and DRT→ separate strands and add second primer→PCR→cDNA clones
How gene expression can be studied using cDNA
Patterns of gene expression are distinct in:
- different cell types
- different developmental stages
- cells grown under different conditions
-normal Vs disease states
The expression of individual genes can be investigated or global changes in gene expression analysed, this is known as transcriptomics
Using DNA microarrays to measure gene expression changes
Commercial arrays are available that include all genes in an organism. They resemble a computer chip the size of a microscope slide. On them the unlabelled genes of one genome are spotted on to form an array. The position of each gene spot is known which means they can be identified.
These gene sequences are actually short fragments of each of the genes like PCR primers, identified for every gene that is known in a particular organism - synthesised and spotted onto these plates
They can be screened in the same way that is done for a Southern blot - hybridisation with a particular gene of interest or a particular population of cDNAs
Using a DNA microarray to measure gene expression changes
E.g. in a tumour
- you would isolate tumour cells and normal cells as a control for comparison
- isolate mRNA from both and compare the cDNA from each
- when you synthesise cDNA you can make them differentially fluorescent by incorporating nucleotides that make them fluoresce.
- incorporated into the reverse transcriptase mix so that the cDNA can be differentiated
- fluorescent cDNA strands are then allowed to anneal to complementary sequences on a commercial array
- unhybridised cDNA is washed off
- genes are hydrolysed to the array and express different fluorescent colours
E.g. red = expressed in tumour cell
Green = expressed in normal cell
Yellow= expression in both types
As location and identity of spots are known this gives you a readout of relative levels of expression of every gene in that organism in both normal and diseased state.
Using microarrays to do transcriptomic studies to compare large populations of mRNA can be replaced (or followed) by a PCR technique
PCR required gene specific primers for each of your genes so usually after a global screening using a microarray you decide on specific genes of interest (usually around 10) e.g. to double check genes that appear to be upregulated in disease state
Done by making cDNA using gene specific primers
End point pcr
We’ve previously looked at end point pcr
The result of setting up a PCR sequence and running it for 30 cycles run in gel electrophoresis for fragment observation.
This tells you that a product is produced and therefore that the gene is present in mRNA however it doesn’t tell you how much product is produced and the amount of PCR product depends on how much template was there originally
