Techniques Flashcards
What are the stages of PCR?
- Sample, primers, free nucleotides and taq polymerase mixed together
- Heated to 96 degrees to denature
- Cooled to 60 degrees to allow annealing of primers
- Heated to 72 degrees - dNTPs and DNA polymerase synthesise new strand, starting at primer
- Cycle repeats approx. 30 times
- DNA separated based on size using agarose gel electrophoresis
What is qPCR used for?
Quantifying the amount of a specific DNA sequence of the expression level of specific mRNA
How is quantities of DNA produced by qPCR measured?
Fluorescent signal e.g. SYBR green
Give an example of a control for qPCR. Why is this necessary?
Take DNA mix and do separate qPCR with primers for gene whose expression we doesn’t think change
e.g. GAPDH
Why can’t we compare the control gene to the testing gene?
Uses different sets of primers
May not be equally efficient
What is NGS used for?
Determining DNA sequence of a large number of different DNA molecules simultaneously
Genome sequencing
cDNA sequencing
What are the stages of NGS?
- Genome fragmented - each fragment amplified
- Fluorescently labelled nucleotides added
- Sequential DNA polymerisation occurs
- Sequence determined by stepwise colour pictures
What are some of the applications of NGS?
Sequence individual genomes
Exome sequencing
What are transgenes?
Genes that are introduced into a cell using transfection and infection
What are the stages of introducing new genes into a cell?
- Purify mRNA from cells
- Turn all mRNA to cDNA using reverse transcriptase
- Amplify cDNA of interest using PCR
- Primers for PCR have additional short sequence with a restriction enzyme site
- cDNA inserted into plasmid downstream of promoter region (can be tagged e.g. GFP)
- Replicate plasmid inside bacteria
- Plasmid introduced to target cell - promoter drives expression of cDNA
What is RNAseq used for?
Quantifying how much mRNA from each gene is present in a sample
What are the stages of RNAseq?
- Purify mRNA from control and test cells
- Reverse transcribe mRNA to cDNA
- Sequence cDNA using NGS
- Quantifies amount of each cDNA and hence mRNA
- After analysis, can create a list of up/downregulated genes for the condition investigated (DAVID)
What are the stages of creating a CRISPR construct?
- Identify 20bp target sequence
- Synthesise sequence. Clone upstream of a sequence that when transcribed to RNA, will bind to Cas9
- sgRNA - small guide RNA with targeting sequence and Cas9 binding sequence
- Once introduced to target cell, guide sequence will momentarily hybridise with target sequence
- Recuits Cas9 and lead to cleavage of target sequence
What are the stages of DNA repair after cleavage by CRISPR?
- Can occur through non-homologous end joining
- Small indels common which disrupt the ORFs - can stop protein function
- Can also use homologous recombination
How can we repair mutated genes with CRISPR?
- Target CRISPR to within mutated sequence and introduce WT DNA as well
- Cell can then use WT DNA for repair (homologous recombination), eliminating the mutation from the genomic DNA
How can we identify PAMs for CRISPR in sequences?
xGG (5’ to 3’) or CCx (3’ to 5’)
