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molecular techniques Flashcards

1
Q

Q: Why identify genes important for cell function?

A

A: To understand which genes are essential for specific cellular roles and to investigate their effects.

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2
Q

_ clones represent the entire DNA in an organism’s genome, including _ regions, _ sequences, and _ DNA.

A

Genomic DNA
noncoding
repetitive
regulatory

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3
Q

_, derived from mRNA, contain mainly _-coding sequences, allowing researchers to study which genes are expressed in specific cells or developmental stages.

A

cDNA clones
protein

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4
Q

Q: What are some techniques used to manipulate gene expression?

A

A: Transfection, conditional knockouts, and siRNA.

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5
Q

Q: What is the goal of transfection?

A

A: To increase the expression of specific genes in cells.

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6
Q

Q: What does a conditional knockout do?

A

A: It selectively removes gene expression in specific conditions.

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7
Q

Q: What is siRNA used for in molecular biology?

A

A: To silence or reduce the expression of target genes.

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8
Q

Q: What is important to consider when choosing molecular cell biology approaches?

A

A: Understanding how and when to apply specific techniques effectively.

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9
Q

Q: What advantage did Taq polymerase provide for the PCR technique?

A

A: Taq polymerase can withstand high temperatures required in PCR, allowing DNA to be amplified without enzyme degradation.

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10
Q

Q: What is the purpose of qPCR?

A

A: To study the amount of a specific mRNA in a cell.

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11
Q

Q: What is the first step in a qPCR experiment?

A

A: Isolate RNA from the cell.

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12
Q

Q: Why is PCR needed in qPCR?

A

A: The amount of RNA is often too small to measure or visualize directly, so PCR is used to amplify it.

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13
Q

Q: What challenge does RNA pose in qPCR?

A

A: DNA polymerase cannot recognize RNA because of structural differences between RNA and DNA.

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14
Q

Q: How is RNA converted into a form that can be used in PCR?

A

A: A DNA copy of the RNA, called cDNA, is made.

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15
Q

Q: Which enzyme is used to convert RNA into cDNA for qPCR?

A

A: Reverse Transcriptase.

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16
Q

Q: What is the purpose of hybridizing mRNA with a poly(T) primer in qPCR?

A

A: To initiate the creation of cDNA by reverse transcription.

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17
Q

Q: What happens to mRNA after the first DNA strand is synthesized in qPCR?

A

A: mRNA is degraded with RNase H, leaving a single cDNA strand.

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18
Q

Q: What is the final product of the cDNA synthesis process in qPCR?

A

A: A double-stranded cDNA copy of the original mRNA.

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19
Q

Q: What are the essential materials needed for a PCR reaction?

A

A: DNA template, primers, nucleotides (G, A, T, C), and DNA polymerase.

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20
Q

Q: Why are primers crucial in PCR?

A

A: They are specific sequences that bind to the target DNA region, enabling selective amplification.

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21
Q

Q: What are the three main steps in a PCR cycle?

A

A: 1) Heat to separate DNA strands, 2) Cool to allow primer binding, 3) DNA synthesis from primers.

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22
Q

Q: How many times is DNA amplified after 40 PCR cycles?

A

A: Approximately 2^(40), resulting in about 1 trillion copies of the target DNA.

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23
Q

Q: How is DNA detected in PCR at the end of the process?

A

A: Using a fluorescent dye that binds specifically to double-stranded DNA.

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24
Q

Q: When is fluorescence measured in qPCR?

A

A: After every PCR cycle to monitor the accumulation of DNA in real time.

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25
Q: What happens to SYBR Green during denaturation in qPCR?
A: SYBR Green is released, and fluorescence decreases as the DNA strands separate.
26
Q: What is the role of SYBR Green in qPCR?
A: SYBR Green fluoresces when bound to double-stranded DNA, indicating DNA synthesis.
27
Q: When does SYBR Green bind to DNA again in qPCR?
A: During the extension phase, when new double-stranded DNA is formed, increasing fluorescence.
28
Q: What is the Ct value in qPCR?
A: The cycle threshold (Ct) is the point where fluorescence reaches a detectable level, used for quantitation.
29
Q: What does it indicate if fewer cycles are needed to reach the Ct value?
A: A higher initial quantity of the target DNA is present in the sample.
30
Q: How is amplification efficiency assessed in qPCR?
A: By plotting a standard curve with serial dilutions and checking for consistent doubling (approximately 3.2 cycles difference per 10-fold dilution).
31
Q: What is a closed-ended approach in molecular biology?
A: A technique like PCR or gene sequencing that uses known primers to detect specific, known sequences.
32
Q: What is an open-ended approach in molecular biology?
A: Techniques like microarrays or DNA/RNA sequencing where the outcome is not predefined, allowing detection of unknown sequences.
33
Q: When would you use a closed-ended approach?
A: When you want to confirm the presence of specific, known sequences in a sample.
34
Q: When would you use an open-ended approach?
A: When exploring a sample without prior knowledge of the sequences, to discover or analyze unknown genetic material.
35
Q: What is the purpose of genetic sequencing in molecular biology?
A: To determine the exact sequence of nucleotides (G, A, T, C) in a DNA segment.
36
Q: What is Sanger sequencing?
A: A DNA sequencing method that uses dideoxynucleotides to terminate DNA strand elongation, allowing the sequence to be determined.
37
Q: What key components are needed for Sanger sequencing?
A: A DNA library, DNA primer, DNA polymerase, and dideoxynucleoside triphosphates (ddNTPs).
38
Q: What is the role of dideoxynucleoside triphosphates (ddNTPs) in Sanger sequencing?
A: They terminate the DNA strand because they lack the 3' OH group needed for chain elongation.
39
Q: How is DNA sequencing read in modern Sanger sequencing?
A: Each nucleotide (G, A, T, or C) is labeled with a different color, and a sequencing machine reads the colors to determine the sequence.
40
Q: What is the difference between a normal deoxyribonucleoside triphosphate (dNTP) and a chain-terminating dideoxynucleoside triphosphate (ddNTP)?
A: dNTPs have a 3' OH group allowing extension, while ddNTPs have a 3' H that prevents further extension.
41
Q: What does PCR stand for?
A: PCR stands for Polymerase Chain Reaction,
42
Q: How are DNA fragments separated in Sanger sequencing?
A: DNA fragments of different lengths are separated by gel electrophoresis, where smaller fragments migrate faster.
43
Reading DNA Sequencing Results
DNA fragments are separated by size in gel. Smallest fragments move fastest, sequence is read from bottom to top.
44
Q: Why do primers targeting exon boundaries help avoid DNA amplification? Q: How do exon-spanning primers prevent DNA contamination?
A: They recognize adjacent exon parts, preventing amplification of genomic DNA. A: By hybridizing only to mRNA sequences where exons are spliced together.
45
Q: What does NFATc1 downregulation indicate?
A: It shows relative expression changes compared to a housekeeping gene.
46
Q: Why are housekeeping genes used in gene expression analysis?
A: They provide a reference for relative expression.
47
Q: What condition is caused by a mutation in Cathepsin C?
A: Juvenile periodontitis.
48
Q: What role does Cathepsin C play in immune response?
A: It is necessary in granulocytes to kill bacteria.
49
Q: What are the symptoms of juvenile periodontitis due to Cathepsin C mutation?
A: Inflammation and tooth loss.
50
Q: How can primers be designed to detect only cDNA and not DNA?
A: By designing primers that either span exon-intron boundaries or flank introns, so only cDNA (without introns) is amplified.
51
Q: What is (bulk) RNA sequencing used for?
A: It's an open-ended approach to analyze differential gene expression between conditions (e.g., healthy vs. diseased samples).
52
Q: List the basic steps in RNA sequencing methods. A: 1. RNA isolation and cDNA synthesis with barcoding 2. 3. Amplification with PCR 4. Sequencing and raw data analysis
Sample and library preparation
53
Q: What is the significance of the HUGO project in DNA sequencing?
A: It aims to determine the total genome sequence and detect genetic mutations.
54
Flashcard 4: Q: What is the purpose of RNA sequencing?
A: It is used for differential gene expression analysis in various conditions (e.g., control vs experimental, healthy vs diseased).
55
Flashcard 5: Q: What are the main steps in RNA sequencing methods?
A: 1) RNA isolation and cDNA synthesis, 2) sample and library preparation, 3) amplification with PCR, 4) sequencing, and 5) raw data analysis.
56
Flashcard 6: Q: What is fibrodysplasia ossificans progressiva (FOP)?
A: A disease where fibrous tissue is progressively replaced by bone, due to a mutation affecting Activin A signaling.
57
Flashcard 8: Q: How does Activin A signaling differ in FOP patients?
A: A mutation (ACVR1[R206H]) alters signaling, leading to abnormal bone formation, as observed in RNA sequencing.
58
Q: How does single-cell RNA sequencing differ from bulk RNA sequencing?
A: Single-cell RNA sequencing analyzes gene expression in individual cells, while bulk sequencing analyzes all cells in a sample together.
59
Q: What can RNA sequencing reveal about pathways?
A: It can show which pathways, such as TGF-beta, BMP, or Activin signaling, are altered in specific conditions like FOP.
60
Q: What do the red dots in RNA sequencing data for FOP represent?
A: Significantly different gene expression when comparing samples with and without Activin A.

Molecular Cell Bio (13 decks)

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