Recombinant DNA repair and molecular techniques part 2(part 2) Flashcards
reverse transcription
Reverse transcriptase
- use RNA as a template to make a complementary strand of cDNA
cDNA synthesis using mRNA
All eukaryotic mRNA have poly A tails
A short poly dT oligonucleotide is annealed to the poly A tail to serves as a primer for enzyme reverse transcriptase.
RT use the mRNA as a template to synthesize the cDNA strand.
cDNA synthesis requires dNTPs
cDNA forms a mRNA/cDNA double stranded duplex.
cDNA in prokaryotes
Prokaryotic mRNA lack a poly A tail.
A random hexamer annealed to the end of mRNA serves as a primer for enzyme reverse transcriptase.
RT uses the mRNA as a template to make the cDNA strand
Synthesis of cDNA requires dNTPs
Real time PCR
With advancement of real time PCR, detection of PCR products can be carried out throughout the amplification process.
Does not use agarose gel
A fluorescent dye binds to newly synthesized double stranded PCR products as they are generated
Fluorescent signal is proportional to the number of PCR products made.
SYBR green based real time PCR
SYBR green dye attach to double stranded DNA
When the DNA denatured the SYBR, green dye floats free.
Extension phase begin as primers anneal
Polymerization is complete again, SYBR green dye binds to the dsDNA product and fluorescents.
Quantifying gene expression using RT PCR
Total RNA is 1st extracted from different cells and tissue samples
RNA extracted from different samples are converted to cDNA using reverse transcriptase.
Real time PCR primers are designed for target genes.
RT-PCR use cDNA as target template
there is a quantitative relationship between amount of starting cDNA sample and amount of PCR product at a given cycle number
Normalized fluorescence to PCR cycle number curve
There will be multiple cycle on x axis, PCR cycle numbers are labelling the cycle threshold.
it is 5 curves joined together, with different plateaus, exponential growth rate is seen in all 5 curves.
As PCR progress, more PCR products are produced, more fluorescence are detected.
How to plot standard curve
From the amount of PCR products at a given cycle, we can find the amount of cDNA.
We then plot a cycle number vs Log (cDNA concentration) on a standard curve.
This standard curve helps us to track the amount of DNA produced through the timing of the cycles.
Expression vectors
Cloning vectors with the ability to encode for transcription and translation of DNA insert.
allows a cloned DNA insert to be translated into a protein inside a bacteria or eukaryotic cells
What are the 7 important parts of expression vectors
- ori
- vector selection marker
- strong promoter
- accommodate DNA insert in multiple cloning site
- direct tagging of gene products
- transcription terminator
- used for producing large amounts of specific proteins
GST-tag-labelling of gene product
creates a fusion product that is a hybrid of the gene product and GST tag
GST-tag affinity matrix with glutathione
2 issues of expression of eukaryotic genes in bacteria
- Protein instability
2. Non-authentic protein
Why non-authentic protein product
Bacteria do not perform PTM as the prokaryotic cells lack the necessary enzymes.
No disulfide bond formation, which are used to stabilize tertiary and prokaryotic structure.
No proteolytic cleavage of inactive precursor protein
DNA microarrays or DNA chips
Study global gene expression of organisms using DNA chips
DNA microarrays rough concept
It can detect and measure which genes are activated and which genes are repressed simultaneously when 2 populations of cells are compared.
Single stranded DNA representing different genes are fixed to a DNA chip
Hybridization with fluorescent dye labelled cDNA
Hybridization scanned with imaging system and analyzed with computer software.
