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mod 1 Flashcards

a brief overview of mod 1 before going into more detail from my notes (68 cards)

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1
Q

What is the natural role of restriction endonucleases?

A

Bacterial defense — cut invading viral DNA.

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2
Q

How are restriction endonucleases named?

A

1st letter = Genus, Next 2 = species, Number = discovery order.

Example: HaeIII = Haemophilus aegyptius, 3rd enzyme.

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3
Q

What are cut sites for restriction endonucleases?

A

Palindromes (e.g., GAATTC / CTTAAG).

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4
Q

What are sticky ends?

A

Staggered cuts, single-stranded overhangs → H-bonds.

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5
Q

What are blunt ends?

A

Straight cut, no overhang → no H-bonding.

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6
Q

What is the purpose of gel electrophoresis?

A

Sort DNA/RNA by size/charge.

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7
Q

What are the steps in gel electrophoresis?

A

Apparatus: Load DNA in polyacrylamide gel, Apply voltage, - charged SDS moves to positive anode.

Sieving: molecules sperate by size with smaller fragments moving faster.

Visualization: 4 different restriction enzyme digestions from 2 vial strains formed, stained with ethidium bromide (to see DNA bands)

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8
Q

How is visualization achieved in gel electrophoresis?

A

Ethidium bromide staining + UV light.

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9
Q

What is the goal of DNA cloning?

A

Isolate and Amplify a DNA sequence using bacteria.

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10
Q

What are the steps in DNA cloning?

A
  1. Isoalte DNA of interest
  2. Cut DNA and cloning vector with restriction endonucleases to produce matching sticky ends
  3. Mix the fragments; complementary sticky ends form hydrogen bonds
  4. DNA ligase forms phosphodiester bond between fragments
  5. Insert into a Host Cell
  6. As the bacteria divide, they replicate the plasmid.
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11
Q

What is a vector in DNA cloning?

A

Carries foreign DNA (e.g., plasmid, virus) into cell -

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12
Q

What are plasmids?

A

Small circular DNA, replicate independently.

restriction enzyme inserts genes into plasmid and trasfects it into mammal cell
THEN drug is added and only cells wiht plasmid will survive

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13
Q

What is a DNA probe?

A

Radiositope/non-radioactively labeleld single-strand DNA → detects/binds to complementary sequence.

carry out hybridization/binding

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14
Q

What types of labels can DNA probes have?

A

Radioactive (32P) or Nonradioactive (fluorescent, biotin)

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15
Q

What are ASOs (Allele-Specific Oligonucleotides)?

A

Designed to bind to specific alleles

(e.g., mutations or polymorphisms)

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16
Q

How do ASOs work?

A
  1. DNA and ASO mix
  2. IF DNA has mutation –> probe binds (hybridization)
  3. If DNA low –> amplified by PCR –> mutation now detectable
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17
Q

What is an example of ASOs usage?

A

Sickle cell (β-globin gene): associated with ozygen trnasport of hemoglobin
βA = normal
βS = mutant

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18
Q

What does ‘S N O W D R O P’ stand for in blotting techniques?

A

Southern = DNA, Northern = RNA, Western = Protein.

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19
Q

What are the steps in Southern/Northern blotting?

A

Gel Electrophoresis: DNA/RNA separated by size.
Transfer: DNA/RNA transferred to nylon or PVDF membrane.
Blocking: Prevents nonspecific binding of probes
(wash with prehybridize solution)
Hybridization: Labelled probes bind to complementary DNA/RNA sequences.
(washing with radioactive, biotin, or fluorescently-labeled DNA probes)
Detection: Using radioactive (@ -80C ) or fluorescent (@ room temp) labels to visualize bond sequences

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20
Q

What is PCR (Polymerase Chain Reaction)?

A

Amplifies specific DNA → generate many copies

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21
Q

What are the ingredients for PCR?

A

Primers (ssDNA), Taq polymerase (heat-stable), dNTPs, Mg²⁺, DNA.

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22
Q

What are the cycles of PCR?

A

Denature: 95°C → DNA strands separate
Anneal: 55–60°C → primers bind to DNA
Extend: 65°C → Taq polymerase adds dNTPs to 3’ end.

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23
Q

What are applications of PCR?

A

Mutation detection, Viral DNA (HIV)/low abundance N.A sequence, Forensics (single hair, blood).

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24
Q

What is Allele-Specific PCR?

A

Detect different DNA alleles @ specific mutation site (wanna find the version someone has)

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25
What are the results for homozygous and heterozygous in Allele-Specific PCR?
Homozygous: 1 band, Heterozygous: 2 bands.
26
What are microarrays?
Analyze thousands of DNA sequences at once to identify 1) Gene VARIATION (mutation, SNPs) 2) Gene EXPRESSION (turn genes "on" / "off")
27
How do microarrays work?
labeled with fluorescent→ exposed to gene chip with DNA spot → fluorescence intensity = expression level. ## Footnote DNA: used to genotype cDNA: used for gene expression
28
What is the use of microarrays?
Identify cancer gene signatures for prognosis or therapy.
29
What is CRISPR-Cas9?
Genome-editing tool that can cut DNA at specific sites to destory virus ## Footnote has adaptive immunity: identifies virus and attacks it via restriction endonucleases
30
Steps of CRISPR-Cas9 in Bacterial Immunity
1) Viral Infection (injected into DNA) 2) Foreign DNA recognition --> cut into small fragments 3) DNA inserted into bacterial genome at CRISPR loci (act as memory of past infections) 4) Guide RNA (gRNA) Production (matches original viral DNA sequence) 5) If virus infects again gRNA bind to viral DNA 6) **Cas9** enzyme unwinds to check if viral DNA is complementary --> cuts DNA
31
What is RNA Biotechnology?
Includes techniques like 1) Northern Blot 2)Quantitative PCR 3) cDNA Microarray
32
What does Northern Blot detect?
detect specific RNA expression/sequence → confirms if gene is 'on'. ## Footnote Use probe
33
what are the purpose of blots
* used for only a few samples to confirm results * used when you want to know about size
34
What is Quantitative PCR (qPCR)?
* mRNA reverse transcibe into → cDNA → PCR * Fluorescence tracks amplification * Measures amount of expression for ONE gene
35
What does qPCR measure?
Gene expression.
36
What is cDNA Microarray?
mRNA → cDNA (via reverse transcriptase) --> tagged with fluorescent dye --> MIX --> place on microarray chip
37
What does more color in cDNA Microarray indicate?
More gene expression. ## Footnote copmaring gene expression between normal and diseased cell
38
What is SDS-PAGE?
Separates proteins by size. | via polyacrylamide gels & SDS detergent
39
What does SDS do in SDS-PAGE?
Detergent that denatures proteins, gives them uniform charge.
40
What is Western Blot used for?
For protein detection. | used after transfer of SDS-PAGE to nitrocellulose or PVDF ## Footnote Use antigen-specific enzyme-labeled antibody →generates “band”
41
What is the process of Western Blot?
After SDS-PAGE, transferred to membrane, Use enzyme-labeled antibodies → generate bands = size + presence.
42
What does ELISA quantify?
Quantify Protein Expression | application: detect immune response ## Footnote ditect protein-protein OR protein-antigen interactions
43
What are the formats of ELISA?
**Direct**: Coated directly with antigen **Sandwich**: Coated with "capture" antibody
44
What is the difference between Western Blot and ELISA?
Western: Qualitative & size, ELISA: Quantitative.
45
What are enzyme assays?
Continuous: Monitors reaction in real time, Discontinuous: Stops reaction to measure product.
46
What techniques can be used for enzyme assays?
Use ELISA, Western, or color changes.
47
Applications of Cloned DNA
1. Expressed to produce proteins 2. Used for other DNA cloning experiments 3. Isolated and analyzed
48
What is Blotting
Transfer of macromolecules, such as nucleic acids or proteins to a solid-phase membranous support.
49
Northern and Southern Blotting Steps
1) **Gel Electrophoresis **(seperate DNA/RNA by size) 2) **Transfer** (DNA/RNA moved to nylon or PVDF membrane) 3) **Blocking** (prevent nonspecific probe binding) 4) **Hybridization** (labelled probs bind to compementary DNA/RNA sequence) 5) **Detection** (use fluorescent/radioactive to visualize)
50
PCR
Amplifies specific DNA sequence
51
The PCR Cycle
**Denaturation**: DNA heated to separate strands (95°C). **Annealing**: Primers bind to single-stranded DNA (55-60°C). **Extension**: DNA polymerase synthesizes complementary strands using dNTPs by adding the 3’ of the DNA primer (65°C).
52
What is the primary application of PCR in detecting low abundance nucleic acid sequences?
Enables detection of viral DNA (e.g., HIV) even in early stages. ## Footnote This is particularly useful during the latency period of infections.
53
What is one key advantage of PCR in comparing normal and mutant genes?
Allows synthesis of enough mutant DNA to directly determine its sequence without cloning. ## Footnote This is crucial for genetic analysis and research.
54
What is the function of mismatched primers in PCR?
Primers with a mismatched 3'-residue will not function under proper PCR conditions. ## Footnote This ensures specificity in amplification.
55
What distinguishes homozygous from heterozygous samples in allele-specific PCR?
Homozygous produces products from matching SNP primer, while heterozygous produces from both allele-specific primers. ## Footnote This is important for genetic variation analysis.
56
What are microarrays used for?
To compare large numbers of samples simultaneously and determine mutation locations or identify multiple SNPs. ## Footnote Useful in genomics and personalized medicine.
57
What is CRISPR-Cas9 primarily used for?
Directly edit genome of cells for specific sequences. ## Footnote This technology has revolutionized genetic engineering since its development.
58
What does the term 'adaptive immunity' refer to in the context of CRISPR?
A defense mechanism found in prokaryotes to protect against viruses by recognizing and attacking them. ## Footnote This system utilizes restriction endonucleases.
59
What is the role of Cas9 in the CRISPR system?
It is an RNA-guided DNA endonuclease that cleaves foreign DNA. ## Footnote Cas9 unwinds DNA to check for complementarity with guide RNA.
60
What is the main purpose of Northern Blot?
To show and detect specific RNA sequences and confirm gene expression. ## Footnote It is performed from isolated mRNA.
61
What is the process involved in quantitative PCR?
mRNA is reverse transcribed into cDNA and fluorescence is measured during the qPCR process. ## Footnote This technique quantifies gene expression levels.
62
What is the purpose of cDNA microarrays?
To analyze gene expression by measuring fluorescence from cDNA generated from mRNA. ## Footnote This helps compare gene expression in different tissues.
63
What is SDS-PAGE used for in protein analysis?
To separate proteins by size using polyacrylamide gels with SDS detergent. ## Footnote SDS maintains proteins in a linear and unfolded state.
64
What is the role of antibodies in protein detection?
They target specific antigens and can be attached to enzymes or fluorescent markers for detection. ## Footnote Antibodies are crucial for diagnostics and research.
65
What is the main difference between Western Blot and ELISA?
Western Blot confirms protein presence and size, while ELISA quantifies protein expression. ## Footnote Both techniques are used for protein analysis but serve different purposes.
66
What are the two main types of enzyme activity assays?
Continuous assays and discontinuous assays. ## Footnote Continuous assays measure activity over time, while discontinuous assays measure products at specific points.
67
Fill in the blank: CRISPR stands for _______.
Clustered Regularly Interspaced Short Palindromic Repeats.
68
True or False: cDNA microarrays can be used for both gene expression analysis and genotyping.
True.

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