cycle 10 Flashcards
process of PCR (reagents, temperatures, steps)
1 - denaturation, 95°C, DNA sample containing the target sequence, heat-stable DNA polymerase, primers, nucleotides, high temp denatures DNA into single strands, breaking h bonds
2 - annealing, 55º, complementary to target DNA sequence, temp lowered to allow primers to anneal (bind) to complementary sequences on single-stranded DNA template
3- synthesizing, 72ºC, heat-stable DNA polymerase (Taq) and nucleotides (dNTPs), temp increased to optimal working temp allowing DNA polymerase to synthesize new DNA strands by extending the primers
how RT-PCR is used to quantify the abundance of mRNA (also known as gene expression levels or transcript levels)
mRNA is extracted, treated with DNase, converted into cDNA (only exons in DNA form) through reverse transcription (using reverse transcriptase)
target cDNA sequence is amplified by traditional PCR
cDNA represents the expressed genes
know the process of reverse transcription
RNA is extracted and isolated
primer is attached
reverse transcriptase is added to reaction mix
synthesizes complementary DNA strand
displaces mRNA strand and generates single-stranded cDNA molecule
remaining enzymes are inactivated and removed
understand how PCR is used for DNA profiling
str analysis - 1 in hundreds of trillions chance that someone else has the same STR sequence
sex determination - XX x band thicker XY one x one y, longer
difference between mRNA and cDNA
mRNA is an RNA molecule that exists in your cells. cDNA does not exist because we don’t have reverse transcriptase and cannot do reverse transcription. cDNA is used for experiments only
process of RT-PCR
RNA is extracted and isolated
only mRNA is targetted (polyA tail)
primer (Oligo(dT) primer - specific for polyA tail, starts synthesis
synthesizes cDNA (exonic sequence derived from mRNA) - represents expressed genes
how is it possible to express a human gene in a bacterial cell
must place the cDNA sequence of the gene into bacterial cell. cDNA only represents exonic sequences so bacteria is able to process the gene
how is it possible for bacterial to synthesize human insulin
RT-PCR is used to make human insulin cDNA which is cloned in plasmid and transformed in bacteria
the mechanism of the adaptive immune system of bacteria
if bacteria fights and survives infection, turns on CRISPR locus. cuts viral DNA and puts in CRISPR locus. when reinfected, CRISPR turns on again
how does the cell repair double stranded breaks
NHEJ (non-homologous end joining) - error prone
HDR (homology-directed repair) - accurate, uses the homologous chromosome to copy over the sequence and repair the damage
how NHEJ and HDR are used by CRISPR-Cas9 to disrupt, correct mutations or insert normal copy of gene
introduce indels, precisely correct mutations
how does base editing work
nCas nicks one strand
dCas (dead) cannot cut
fuse Cas9 with other proteins to change base
UGI inhibits uracil DNA glycosylation (prevents fixing)
cut is in other strand, mismatch repair system on
thinks mutation is in cut strand
DNA replication - base changed
know how CRISPR is being used to advance immunotherapy
CRISPR edits T-cells, edited t-cell does not express PD-1, recognizes cancer cells
