Cycle 10: DNA Technologies Flashcards
understand the mechanism of the adaptive immune system of bacteria.
CRISPR-Cas system. Bacteria remember and defend against specific viral infections.
1) CRISPR Loci: CRISPR region with DNA sequence seperated by spacer derived from past infections.
2) Cas Proteins: enzymes targets and cut viral DNA
what is the process of the adaptive immune system in bacteria
1) adaptation: When virus infects DNA, a piece of that DNA is captured and inserted into CRISPR locus as a new spacer
2) Expression: CRISPR locus is transcribed into RNA which guides Cas to viral DNA.
3) Interference: Cas cuts viral DNA, prevents virus from being replicated
How does the cell repair double-stranded breaks in cell
NHEJ AND HRD
Why is NHEJ error prone and HRD is not error prone
NHEJ: Directly ligates broken DNA ends without using a template. Because there is no template NHEJ might mutations or deletions at the repair site
HDR: Uses homologous sequence as a template to repair the break accurately. It uses a template hence it has a lower chance of making a mistake.
How NHEJ and HDR Are Used by CRISPR-Cas9
NHEJ: disrupt genes making some harmful genes not work
HDR: correct mutations or insert new sequences
How does base Editing work? advantage and application
Mechanism: Bases editing uses Cas fused with an enzyme to modify specific DNA bases
Advantage: It does not create double stranded breaks making it more precise.
Application: can correct single point mutations.
How is CRISPR advancing immunotherapy
PD-1 KO: use CRISPR on T cells to make immune system attack cancer
Understand the process of PCR (Reagents)
DNA template: The DNA sequence to be amplified.
Primers: Short DNA sequences (Forward and Reverse) that bind to the target DNA region.
dNTPs (deoxyribonucleotide triphosphates): The building blocks for the new DNA strands.
DNA polymerase: Enzyme that synthesizes the new DNA strands (e.g., Taq polymerase).
MgCl₂: Magnesium chloride, a cofactor for DNA polymerase.
understand PCR (Temperature)
Denaturation: ~95°C - Separates the DNA strands.
Annealing: ~50-65°C - Primers bind to the target DNA sequence.
Extension: ~72°C - DNA polymerase extends the primers, synthesizing new DNA strands.
PCR steps
Denaturation: DNA is heated to separate the double-stranded DNA into single strands.
Annealing: Primers bind to the complementary sequences on the single-stranded DNA.
Extension: DNA polymerase extends the primers, creating new DNA strands.
Repeat: The cycle is repeated multiple times to amplify the target DNA exponentially.
How is RT-PCR used to quantify the abundance of mRNA
process: mRNA becomes complementary DNA then quantified VIA PCR amplification
Explain process of reverse transcription
1) The oligo (dt) primer binds to poly-A tail of mRNA.
2) Reverse transcriptase synthesizes cDNA from the mRNA template
How is PCR used for DNA profiling (STR regions and sex determination)
STR regions: Highly polymorphic regions in the genome used for DNA profiling.
PCR amplifies STR regions, and the number of repeats varies between individuals.
Sex determination: PCR can amplify specific regions of the Y chromosome to determine sex (e.g., presence or absence of Y chromosome markers).
How to read electropherogram
After PCR, the amplified DNA fragments are separated by size using electrophoresis.
The results are displayed as peaks on an electropherogram, where each peak represents a DNA fragment of a specific size.
Difference btwn mRNA and cDNA
mRNA: exists in ur cells
cDNA: DNE in cells, only used in experiments. cuz we dont have reverse transcriptase
What are the components of RT-PCR
mRNA: The RNA molecule to be quantified.
Reverse Transcriptase: Enzyme that synthesizes cDNA from mRNA.
Oligo(dT) Primer: Binds to the poly-A tail of mRNA to ensure only mRNA is reverse transcribed.
dNTPs: Building blocks for cDNA synthesis.
Mechanism of RT-PCR
mRNA is reverse transcribed into cDNA.
The cDNA is then amplified using PCR to quantify the original mRNA levels.
Purpose: RT-PCR is used to determine mRNA levels (gene expression levels or transcript levels).
How to express human genes in a bacteria
cDNA Synthesis: RT-PCR is used to create cDNA from human mRNA.
Cloning: The cDNA is inserted into a plasmid vector.
Transformation: The plasmid is introduced into bacterial cells.
Expression: Bacteria use the cDNA to produce human proteins (e.g., insulin) because cDNA lacks introns, which bacteria cannot process.
How can bacteria make human insulin?
cDNA Synthesis: RT-PCR is used to create cDNA from human insulin mRNA.
Cloning: The insulin cDNA is inserted into a plasmid vector.
Transformation: The plasmid is introduced into bacterial cells.
Expression: Bacteria produce human insulin using the cDNA template.
Why cDNA?:
cDNA lacks introns, which bacteria cannot process, making it ideal for expressing human genes in bacterial cells
