8. Recombinant DNA and biotech Flashcards
What is recombinant DNA?
DNA fragments can be rejoined by DNA ligase. Recombinant DNA is any two sequences of DNA that are ligated together.
What’s going on here?
The restriction enzyme EcoRI cuts DNA into smaller pieces (at GAATTC site)
These two strands are ligated together to make recombinant DNA.
What are plasmids used for?
Plasmids are small circular DNA. Plasmids are often used as cloning vectors.
You cut genes of interest and ‘paste them into plasmid
Explain the process here.
DNA fragment of interest (to be cloned) is inserted into antibiotic resistant plasmid. The plasmids are placed into E coli (bacteria) solution. The solution is then cultured on a plate that has antibiotic and nutrients. The bacteria cells that didn’t take in plasmids die. The mechanism of bacteria to multiply helps clone the DNA fragment.
What is the goal of recombinant DNA?
One goal of recombinant DNA technology is to clone a particular gene, either for analysis or to produce its protein product in quantity.
Bacteria, yeasts, and cultured plant cells are commonly used as hosts for recombinant DNA.
What’s the difference between genomic library and cDNA library?
Genomic library is made through recombinant DNA tech starting with genomic DNA
cDNA library is made through recombinant DNA tech starting with mRNAs undergoing reverse transcriptase
How do you carry out DNA sequencing?
DNA sequencing is the way we find out the order of nucleobases in DNA
- Use PCR to amplify sample
- Use fluorescent dNTP (each base with a different colour) but modifies so once added it stops polymerization
- Added nucleotide is detected by a camera
- Repeat cycle about 100-300 times
What is this picture showing?
How DNA fragments (‘reads’) are arranged to get a single assembled sequence.
What does this picture show?
RNA sequencing
RNA is fragmented then reverse transcribed to make cDNA. Then the cDNA is sequenced. From this we can determine the sequence and the quantity of RNA, which corresponds to the sequence/quanitity of cDNA
# of particular reads = a measure of copies of mRNA (cf Ct in qPCR)
What’s transcriptome?
- Transcriptome refers to gene expression. The transcriptome is the complete set of RNA transcripts in a sample at a particular time
- A snapshot of all mRNA in a cell.
- Proteome = “all” proteins
What do they spot colours’ mean in microarray?
What’s happening here?
Transcription and translation
What does downregulation and upregulation mean?
Downregulation refers to a decrease in production of a specific cellular component, whereas upregulation refers to an increase. These changes occur in response to stimuli and can result from a wide range of processes. The components that are referred to as being up- or downregulated may be transcribed RNA or translated proteins.
What’s a volcano plot?
- Easy way to visualize the entire set of gene
expression data - Plots p value (statistical significance) vs.
fold change - Comparing gene expression from RNA seq is RELATIVE because of fold change ratio and PCR amplification
What’s a heat map?
- good for comparing expression of select genes we know we are interested in
- each column indicates sample, and each row represents a gene. The colour of each cell indicates the magnitude of expression of the gene in the sample.
What is this?
tRNA molecule
What’s happening here?
Enzyme charging the tRNA. It will deliver the appropriate amino acid during peptide chain elongation.
What is this?
A ribosome, with three sites for tRNA binding.
tRNA and mRNA will only interact at P and A sites.
How are amino acids added to polypeptide chain?
- Methionine on tRNA binds to mRNA at “P site”
- Second amino acid brought into “A-site”
- Previous a.a. is added to new one.
- old tRNA is kicked off, tRNA with chain moves to P site, new cycle starts
How is translation terminated?
When a stop codon enters the “A” site, a release factor is attached. The polypeptide is disconnected and mRNA and ribosome separate.
How does CRISPR work?
It is a technology to edit genes.
Cas9 works with guide RNA, which guides it to the target DNA. It enables cutting of DNA at desired sequence. (Cas9 is a bacterial enzyme that cleaves both strands of DNA)
The harder step is repairing the DNA. There is imperfect repair.
What are two ways to block mRNA translation?
- With miRNA or siRNA
2. Antisense RNA
