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Winter 2023 - Biology > Cycle 10: DNA Technologies > Flashcards

Cycle 10: DNA Technologies Flashcards

1
Q

What is PCR? What is it based on?

A

Polymerase Chain Reaction
- take target section of genome and amplifying it (make billions of copies)
- difficult to do experiments with small amounts of DNA
- based on DNA replication

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2
Q

What is the process of PCR (reagents, temperatures, steps)?

A

reagents: parent DNA, forward and reverse DNA primers, DNA polymerase (not WT) Taq (isolated from thermophyll), dNTPs (nucleotides), MgCl2 (cofactor for Taq poly)

  1. denaturing 95C
    - double helix unbinds
  2. annealing 55C
    - slowly cools down
    - binding of forward and reverse primers on each
  3. synthesizing 72C
    - optimal Tag temp
    - dNTPs added
  4. repeat to amplify
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3
Q

What is multiplex PCR?

A
  • primers flank target region
  • multiple primer pairs flank target regions in same test tube
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4
Q

How is PCR is used for DNA profiling?

A
  • STR (short tandem repeats) are sequences of DNA between genes (non-coding)
  • everyone has same STR1 and STR2 but different number of repeats
  • must analyze 13 STRs (in autosomal chromosomes) to know which person bc highly polymorphic (many versions)
  • each person gets 2 chromosomes (one from each parents) and number of repeats in each chromosome can be same or different: (STR loci num rep 1, num rep 2)
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5
Q

How is gel electrophoresis used to analyze PCR products?

A

inject each samples after extracting gene on a gel

less repeats = travel further

two band appear if different number of repeats in same STR loci of diff chromosome

one thick band if same different of repeats in same STR loci of diff chromosome

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6
Q

What is AMEL? How can it be used for sex determination?

A

gene on both sex chromosome that codes for protein that maintain enamel

X AMEL gene has deletion so Y AMEL is longer

for XX –> thicker band on gel
for XY –> thin band at same length as XX and thin band above XX

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7
Q

How can you read an electropherogram?

A
  • bands corresponds to peaks
  • large peak means homozygous for number of repeats of STR or XX
  • compared to standard
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8
Q

What is reverse transcription? What is it used for?

A

used to make cDNA from mRNA through reverse transcription
- measure gene expression (how much mRNA in cell)
- DNA sequencing
- cloning
- diagnosing disease
- pharmacogenetic
- DNA profiling (crime scene analysis or paternity testing)

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9
Q

What does mRNA abundance mean?

A

gene expression/transcript levels

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10
Q

How is RT-PCR used to quantify the abundance (amount) of mRNA?

A

Reverse Transcription PCR
- mRNA reverse transcribed to cDNA (complementary DNA) with reverse transcriptase (isolated from retroviruses)
–> cDNA only has exotic sequence bc it is made from mRNA
–> cDNA represents expressed genes

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11
Q

What is the process of RT-PCR? Components of reverse transcription process?

A
  1. lyse cells and add chemicals to break them into aqueous phase: RNA, interphase: DNA, and organic phase: proteins/lipids
  2. extract all RNA (mRNA and non coding RNA)
  3. target polyA tail with an Oligo dT primer complementary to polyA tail (TTTT)
  4. need Oligo dT, dNTPs and reverse transcriptase to create new strand
  5. reverse transcriptase has DNA poly activity and RNase activity: degrades RNA from RNA-DNA hybrid so only DNA present
  6. DNA synthesis to have double stranded DNA
  7. PCR
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12
Q

What is the difference between mRNA and cDNA?

A

mRNA is an RNA molecule that exists in your cells

cDNA doesn’t exist in your cells because we don’t have reverse transcriptase and can’t do reverse transcription
–> cDNA is only used for experiments

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13
Q

What is oligodT?

A

a series of TTTTT that binds to the polyA tail

primer used to select mRNA via polyA tail

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14
Q

How is it possible to express a human gene in a bacterial cell?

A

you must place the cDNA (NOT genomic DNA) sequence of the gene into the bacterial cell

The cDNA only represents exonic sequences (doesn’t have introns) so the bacteria is able to process the gene.

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15
Q

How is is bacteria used to synthesize human insulin?

A
  1. cDNA put into bacteria bc bacteria is fast replication, but can’t handle introns in pre-mRNA
  2. mRNA of human insulin –> convert to cDNA (RT) make many copies (PCR)
  3. put copies of cDNA into plasmid of bacteria
  4. lyse cell (break up cell), put antibacteria as bait to take out human insulin
  5. wash antibody out for purified insulin
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16
Q

What is the mechanism of the adaptive immune system of bacteria?

A

Cas-9 knows where to make cut based on guide RNA which brings Cos 9 to target sequence on DNA

Immunization/Acquisition (1st Infection)
1. viral genome injected into cell

  1. cas-1 and cas-2 transcribed and translated and these proteins cut the viral DNA and put in in the CRISPR locus of bacterial genome
  2. locus has pattern of palindromic repeats and spacers (segments of viral genome sequences)

Defense/Resistance (2nd Infection)
1. viral genome injected

  1. entire CRISPR locus gets transcribed making pre-crispr RNA and tracer RNA which gets hybridized onto pre-crRNA
  2. RNase cuts pre-crRNA separating each trcrRNA with one spacer each (cr-RNA + tracer-RNA hybrid)
  3. cr-RNA + tracer-RNA hybrid (guide) binds to Cas9 forming ribonucleoprotein complex = CRISPR-Cas9
  4. CRISPR-Cas9 cuts injected viral DNA
17
Q

How was Cas-9 exploited?

A
  • took Cas-9 outside of bacteria, designed other gRNA and put in another bacteria to see if it still cuts and it did! (based on gel bands expected for where Cas-9 would cut)
18
Q

What is Cas?

A

CRISPR association protein - nuclease: protein that cuts nucleotides (DNA) makes double stranded break

19
Q

What is gRNA?

A

guide RNA - complementary to target

– linker loop between cr-RNA and tracr-RNA

doesn’t exist in natural system

20
Q

What is CRISPR? Why is CRISPR important?

A

clustered regularly interspaced short palindromic repeats (important because it is precise)

locus in bacteria where memory of pathogens/virus is stored

21
Q

How does the cell repair double-stranded breaks in cell? What is the difference between the 2 methods?

A

NHEJ and HRD

NHEJ is error prone (deletion, insertion, inversion)

HDR (homology directed repair) make a lot of homologous segment of DNA accessible that is complementary target to copy over the sequence and repair the damage, so not error prone
–> homologous segment = donor DNA

22
Q

How are both NHEJ and HDR used by CRISPR-Cas9 to disrupt, correct mutations or insert normal copy of gene?

A

CRISPR needs PAM: protospacer adjacent motif (NGG)

  1. Cas9 protein cuts double strand break (scientist provides PAM and guide RNA)

want to disrupt gene? don’t provide donor DNA – allow NHEJ

want custom gene? want small correction of mutation? provide lab made donor DNA

23
Q

How does base editing work? How is Cas9 manipulated?

A

more accurate for single point mutation

manipulate Cas 9! - d/nCas (dead/nick) - cut one strand, not both
add Cytidine deaminase
add UGI (uracil glycosylase inhibitor)

start with CG:
1. cytidine deaminase coverts C to U

  1. cells usually trigger uracil glycoslyase to change U to back to C but inhibitor stops enzyme activity
  2. nick is on other strand (not one that has been changed from C to U)
  3. force G to change to A through mismatch repair
  4. DNA replication or repair causes TA
24
Q

How is CRISPR being used to advance immunotherapy?

A
  1. take T-cell from bone marrow of patient with lung cancer
  2. take out PD-1 by targeting promotor region that bind to PD-1 specific transcription factors
  3. CRISPR-Cas9 + gRNA through plasmid into T-cell through electroporation
  4. gRNA guides CRISPR-Cas9 to make double strand break for NHEJ so that there is a mutation in the promotor region that relates to PD-1
  5. Edited T-cell will target cancer cells
25
Q

What is PD-1?

A

protein that protects tissues from autoimmune attack

facilitates chronic infection and tumour progression (allows progression)

target cancer cells but not normal cells

Winter 2023 - Biology (11 decks)

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