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Molecular Cell Bio > molecular techniques > Flashcards

molecular techniques Flashcards

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Biology 101 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

Q: What happens to SYBR Green during denaturation in qPCR?

A

A: SYBR Green is released, and fluorescence decreases as the DNA strands separate.

26
Q

Q: What is the role of SYBR Green in qPCR?

A

A: SYBR Green fluoresces when bound to double-stranded DNA, indicating DNA synthesis.

27
Q

Q: When does SYBR Green bind to DNA again in qPCR?

A

A: During the extension phase, when new double-stranded DNA is formed, increasing fluorescence.

28
Q

Q: What is the Ct value in qPCR?

A

A: The cycle threshold (Ct) is the point where fluorescence reaches a detectable level, used for quantitation.

29
Q

Q: What does it indicate if fewer cycles are needed to reach the Ct value?

A

A: A higher initial quantity of the target DNA is present in the sample.

30
Q

Q: How is amplification efficiency assessed in qPCR?

A

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

Q: What is a closed-ended approach in molecular biology?

A

A: A technique like PCR or gene sequencing that uses known primers to detect specific, known sequences.

32
Q

Q: What is an open-ended approach in molecular biology?

A

A: Techniques like microarrays or DNA/RNA sequencing where the outcome is not predefined, allowing detection of unknown sequences.

33
Q

Q: When would you use a closed-ended approach?

A

A: When you want to confirm the presence of specific, known sequences in a sample.

34
Q

Q: When would you use an open-ended approach?

A

A: When exploring a sample without prior knowledge of the sequences, to discover or analyze unknown genetic material.

35
Q

Q: What is the purpose of genetic sequencing in molecular biology?

A

A: To determine the exact sequence of nucleotides (G, A, T, C) in a DNA segment.

36
Q

Q: What is Sanger sequencing?

A

A: A DNA sequencing method that uses dideoxynucleotides to terminate DNA strand elongation, allowing the sequence to be determined.

37
Q

Q: What key components are needed for Sanger sequencing?

A

A: A DNA library, DNA primer, DNA polymerase, and dideoxynucleoside triphosphates (ddNTPs).

38
Q

Q: What is the role of dideoxynucleoside triphosphates (ddNTPs) in Sanger sequencing?

A

A: They terminate the DNA strand because they lack the 3’ OH group needed for chain elongation.

39
Q

Q: How is DNA sequencing read in modern Sanger sequencing?

A

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

Q: What is the difference between a normal deoxyribonucleoside triphosphate (dNTP) and a chain-terminating dideoxynucleoside triphosphate (ddNTP)?

A

A: dNTPs have a 3’ OH group allowing extension, while ddNTPs have a 3’ H that prevents further extension.

41
Q

Q: What does PCR stand for?

A

A: PCR stands for Polymerase Chain Reaction,

42
Q

Q: How are DNA fragments separated in Sanger sequencing?

A

A: DNA fragments of different lengths are separated by gel electrophoresis, where smaller fragments migrate faster.

43
Q

Reading DNA Sequencing Results

A

DNA fragments are separated by size in gel.
Smallest fragments move fastest, sequence is read from bottom to top.

44
Q

Q: Why do primers targeting exon boundaries help avoid DNA amplification?
Q: How do exon-spanning primers prevent DNA contamination?

A

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

Q: What does NFATc1 downregulation indicate?

A

A: It shows relative expression changes compared to a housekeeping gene.

46
Q

Q: Why are housekeeping genes used in gene expression analysis?

A

A: They provide a reference for relative expression.

47
Q

Q: What condition is caused by a mutation in Cathepsin C?

A

A: Juvenile periodontitis.

48
Q

Q: What role does Cathepsin C play in immune response?

A

A: It is necessary in granulocytes to kill bacteria.

49
Q

Q: What are the symptoms of juvenile periodontitis due to Cathepsin C mutation?

A

A: Inflammation and tooth loss.

50
Q

Q: How can primers be designed to detect only cDNA and not DNA?

A

A: By designing primers that either span exon-intron boundaries or flank introns, so only cDNA (without introns) is amplified.

51
Q

Q: What is (bulk) RNA sequencing used for?

A

A: It’s an open-ended approach to analyze differential gene expression between conditions (e.g., healthy vs. diseased samples).

52
Q

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

A

Sample and library preparation

53
Q

Q: What is the significance of the HUGO project in DNA sequencing?

A

A: It aims to determine the total genome sequence and detect genetic mutations.

54
Q

Flashcard 4: Q: What is the purpose of RNA sequencing?

A

A: It is used for differential gene expression analysis in various conditions (e.g., control vs experimental, healthy vs diseased).

55
Q

Flashcard 5: Q: What are the main steps in RNA sequencing methods?

A

A: 1) RNA isolation and cDNA synthesis, 2) sample and library preparation, 3) amplification with PCR, 4) sequencing, and 5) raw data analysis.

56
Q

Flashcard 6: Q: What is fibrodysplasia ossificans progressiva (FOP)?

A

A: A disease where fibrous tissue is progressively replaced by bone, due to a mutation affecting Activin A signaling.

57
Q

Flashcard 8: Q: How does Activin A signaling differ in FOP patients?

A

A: A mutation (ACVR1[R206H]) alters signaling, leading to abnormal bone formation, as observed in RNA sequencing.

58
Q

Q: How does single-cell RNA sequencing differ from bulk RNA sequencing?

A

A: Single-cell RNA sequencing analyzes gene expression in individual cells, while bulk sequencing analyzes all cells in a sample together.

59
Q

Q: What can RNA sequencing reveal about pathways?

A

A: It can show which pathways, such as TGF-beta, BMP, or Activin signaling, are altered in specific conditions like FOP.

60
Q

Q: What do the red dots in RNA sequencing data for FOP represent?

A

A: Significantly different gene expression when comparing samples with and without Activin A.

Molecular Cell Bio (10 decks)

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