Lecture 21 Flashcards
Can you explain the process of PCR, including the role of the primers, taq polymerase and what happens at the three different temperatures?
The DNA strand is heated to 98 degrees to separate strands. It is cooled to 58 degrees to allow primers to bind to the target single strands. It is then heated to 72 degrees to allow taq polymerase to synthesise the new strand.
What is a cDNA?
cDNA is a complementary DNA that is synthesised from mRNA.
If you have all the cDNAs that can be made from an organism, do you have its entire genome
sequence?
No, just the exome of the organism.
What is RT‐PCR? What research question is it trying to answer?
RT-PCR is reverse transcriptase PCR. It is where a piece of mRNA undergoes reverse transcriptase to produce cDNA. The cDNA undergoes PCR to become amplify. (duplex gene)
What is qPCR?
qPCR (quantitive PCR) used to detect the specific sequence of DNA produce by PCR using fluorescent probes and the level of expression. Probe has specific sequence that is able to bind to target DNA but also allows it to double on itself to form a hairpin so that the quenching molecules is prevents the flourophore from lighting up.
How is a Western blot run? What research question does it answer? How does it differ to an
RT‐PCR?
- Run protein on an SDS-polyacylamide gel (SDS - causes unfolding so protein runs based on size)
- Results transferred on polymer sheet
- Antibodies (1’ and 2’ - specific to target protein) are added with enzyme (chemiluminescence) to identify target protein.
- Target protein is compared with RNA data base.
What is RNA‐seq?
RNA seq is the sequencing of a whole transcriptome (all genes that are expressed at a certain time) in Next Gen sequencing and compared to data base to identify gene.
What components do you need to clone a piece of DNA?
- the specific DNA
- Restriction enzymes
- Bacteria cell
- Plasmid vector
What sequences need to be on a plasmid? What additional sequence does the plasmid need
if you want the cloned gene to be expressed?
Plasmid must have an origin of replication, a gene that makes it antibiotic resistance, promoter (bacteria) and sites that are recognised by restriction enzymes.
Can a gene from genomic DNA be cloned into a plasmid to express the protein?
YES.
What does a restriction enzyme do (in molecular cloning) and how is it used?
It cuts the plasmids, recognising short palindromes in the sequence and producing sticky ends so that the gene may be inserted.
What is used to join the cut ends together in molecular cloning?
DNA ligase.
What does the transformation of bacteria allow us to achieve?
The cloning of specific genes in the genome as well as the expression of certain genes.
What is the value of using a model organism?
Allows for testing of certain products or observation. Medical research, fundament processes
What is tissue culture?
Tissue culture is the growth of tissue/cells from organism externally.
