Lec7 | qPCT and RT-PCR

Question 1

What is the purpose of PCR in biotechnology?

Answer 1

PCR is a process used for the amplification of target DNA.

Question 2

How is the progress of Real-Time PCR monitored?

Answer 2

The progress of Real-Time PCR is monitored by quantifying product accumulation after each cycle, in real-time.

Question 3

What does qPCR stand for, and what does it achieve?

Answer 3

qPCR stands for quantitative PCR, and it is used to determine the amount of target sequence or gene present in a sample.

Question 4

What are the initial materials required for qPCR analysis?

Answer 4

The initial materials required for qPCR are isolated DNA or RNA samples.

Question 5

What occurs immediately after the sample isolation phase in qPCR?

Answer 5

After sample isolation, the integrity of the sample is analyzed.

Question 6

What devices are required for data analysis in qPCR after the qPCR assay?

Answer 6

Data analysis in qPCR requires a Thermal Cycler and an Optical Module for detection.

Question 7

What is unique about the detection methods used in qPCR?

Answer 7

All detection methods in qPCR are based on the emission of fluorescence, but they differ in their underlying chemistry.

Question 8

What does SYBR Green bind to in a qPCR reaction?

Answer 8

SYBR Green binds non-specifically to double-stranded DNA as it is generated during a qPCR reaction.

Question 9

How does SYBR Green contribute to fluorescence detection in qPCR?

Answer 9

SYBR Green emits a fluorescent signal upon intercalating with newly synthesized double-stranded DNA in a qPCR reaction.

Question 10

How is fluorescence generated when using the TaqMan probe detection method?

Answer 10

During the extension phase of PCR, the DNA polymerase cleaves the TaqMan probe, separating the reporter from the quencher and allowing the reporter to emit a fluorescent signal.

Question 11

What are the three phases of the qPCR quantitation curve?

Answer 11

The three phases are the initiation, exponential, and plateau phases.

Question 12

What does the threshold line represent in qPCR analysis?

Answer 12

The threshold line represents the maximum level of fluorescence considered as background.

Question 13

How do users set the threshold line in qPCR analysis?

Answer 13

The threshold line is set automatically by the PCR instrument, or manually by the user.

Question 14

What does the baseline in a qPCR represent?

Answer 14

The baseline represents a successful negative control where no fluorescence is detected.

Question 15

How should negative controls appear in relation to the threshold line?

Answer 15

Negative controls should always appear below the threshold line.

Question 16

What does the Cycle Threshold (Ct) indicate in qPCR?

Answer 16

The Cycle Threshold indicates the cycle number at which the fluorescent signal of a PCR reaction reaches the threshold line.

Question 17

What is the relationship between Ct values and the abundance of the target sequence?

Answer 17

Samples with low Ct values indicate a high abundance of the target sequence, while high Ct values indicate a low abundance of the target sequence.

Question 18

What role does qPCR play in biopharmaceutical manufacturing quality control?

Answer 18

qPCR is used in biopharmaceutical manufacturing to monitor the presence of host cell DNA or RNA.

Question 19

Why is viral safety testing vital for biopharmaceutical products?

Answer 19

Viral safety testing is vital to ensure that biopharmaceutical products are free from viral contaminants.

Question 20

How does qPCR assist in pharmacokinetic and pharmacodynamic studies?

Answer 20

qPCR helps in understanding drug efficacy, distribution, and metabolism in pharmacokinetic and pharmacodynamic studies.

Question 21

How is qPCR used in preclinical research and drug development?

Answer 21

qPCR is used to evaluate the effects of potential drug candidates on specific target genes.

Question 22

What do toxicology studies assess using qPCR?

Answer 22

Toxicology studies use qPCR to assess the effects of pharmaceuticals on gene expression in various tissues and cell types, aiding in identifying potential toxic effects.

Question 23

What is the role of reverse transcriptase in RT-PCR?

Answer 23

Reverse transcriptase is used to synthesize complementary DNA (cDNA) from an RNA template in RT-PCR.

Question 24

What is complementary DNA (cDNA) in the context of RT-PCR?

Answer 24

Complementary DNA (cDNA) is a single-stranded DNA molecule synthesized from an mRNA template using reverse transcriptase.

Question 25

What is the central dogma illustrated with regard to RT-PCR?

Answer 25

The central dogma, in this context, illustrates how reverse transcription synthesizes DNA from RNA, which is the reverse of the usual transcription process.

Question 26

What is the first step in cDNA synthesis?

Answer 26

The first step in cDNA synthesis is the isolation of the RNA of interest from the biological sample.

Question 27

From where must RNA molecules be isolated for cDNA synthesis to be relevant?

Answer 27

RNA molecules must be isolated from the specific cell type that expresses the protein of interest.

Question 28

What is necessary to start the experiment for cloning the human insulin gene?

Answer 28

To clone the human insulin gene, it is necessary to start with human pancreatic islet cells that are actively expressing the insulin gene.

Question 29

hat is the process involved after harvesting the target cells for RNA isolation?

Answer 29

After harvesting, the target cells are lysed to isolate RNA.

Question 30

Why is RNA considered chemically unstable compared to DNA?

Answer 30

RNA is considered chemically unstable because it is rapidly degraded by ribonucleases (RNase) in the cell.

Question 31

How is mRNA separated from other cellular components during RNA isolation?

Answer 31

mRNA is separated from other cellular components using methods like Oligo d(T) Affinity Chromatography, which takes advantage of the poly A tail of mRNA molecules.

Question 32

What does the total RNA fraction contain after separation from cellular components?

Answer 32

The total RNA fraction contains all four types of RNA.

Question 33

How is the poly A tail utilized in mRNA enrichment?

Answer 33

The poly A tail is utilized by binding to the Oligo d(T) in Affinity Chromatography columns to enrich mRNA from the total RNA population.

Question 34

What is checked when quantifying RNA samples using a spectrophotometer?

Answer 34

The RNA sample is checked for purity and integrity to ensure it is free of genomic DNA contamination and RNase activity.

Question 35

What do the ratios 260/280 and 260/230 indicate about the RNA sample?

Answer 35

The 260/280 ratio indicates nucleic acid purity, with values around 2.0 for pure RNA, while the 260/230 ratio indicates the presence of chemical contaminants.

Question 36

What is the function of random primers in reverse transcription?

Answer 36

Random primers bind at different locations throughout the mRNA, amplifying most RNA species, including degraded RNA and viral genomes.

Question 37

How do oligo (dT) primers work in reverse transcription?

Answer 37

Oligo (dT) primers bind specifically to the poly-A tail of mRNA, amplifying the entire mRNA into cDNA.

Question 38

How long is the reaction mixture incubated to synthesize cDNA from the RNA template?

Answer 38

The reaction mixture is typically incubated for 30-60 minutes to allow the enzyme to synthesize cDNA from the RNA template.

Question 39

How is the DNA:RNA heteroduplex removed after cDNA synthesis?

Answer 39

The DNA:RNA heteroduplex is removed either by the RNase H enzyme or by alkaline treatment, both of which degrade the RNA strand.

Question 40

What techniques are used to assess the quality of cDNA after synthesis?

Answer 40

The quality of cDNA is assessed using techniques such as gel electrophoresis or spectrophotometry.

Question 41

What does an A260/A280 ratio of around 1.8 indicate for DNA?

Answer 41

An A260/A280 ratio of around 1.8 indicates pure DNA, while lower ratios may suggest the presence of contaminant proteins.

Question 42

What is a cDNA library in the context of the transcriptome of an organism?

Answer 42

A cDNA library is a collection where mRNA from different sources are reverse transcribed, cloned, and stored for further study.

Question 43

What are two applications of creating and using cDNA libraries?

Answer 43

cDNA libraries enable eukaryotic genes to be expressed in prokaryotes and allow the development of shareable databases.

Question 44

What is a key difference in origin between cDNA and gDNA?

Answer 44

cDNA is derived from mRNA, while gDNA is the complete set of DNA found in an organism’s nucleus or organelles.

Question 45

What is a key difference in stability between cDNA and gDNA?

Answer 45

cDNA is generally considered more stable for storage and study than RNA, and gDNA can be stable when properly stored, although both are generally less stable than intact RNA in living cells due to degradation mechanisms.

Question 46

How does the content differ between cDNA and gDNA?

Answer 46

cDNA contains only transcribed gene sequences without introns, whereas gDNA includes both coding sequences (exons) and non-coding sequences (introns).

Question 47

What is the primary functional difference between cDNA and gDNA?

Answer 47

cDNA primarily functions to capture expressed genes in a form that can be easily studied, while gDNA functions as the blueprint for all genetic instructions within a cell.

Question 48

How does the size of cDNA compare to that of gDNA?

Answer 48

cDNA molecules are generally shorter than the corresponding gDNA since they lack introns.

Question 49

Which PCR technique is best suited for detecting a specific antibiotic resistance gene in bacteria causing hospital infections?

Answer 49

PCR is best suited for detecting a specific antibiotic resistance gene because it can amplify and identify a known target DNA sequence.

Question 50

What PCR technique should be used to study gene expression changes of inflammatory markers like TNF-α and IL-6 after drug administration?

Answer 50

RT-qPCR should be used to study gene expression changes of inflammatory markers as it allows for the quantification of mRNA levels.

Question 51

If a researcher is investigating the viral load of a newly discovered virus, which PCR technique is most appropriate?

Answer 51

qPCR is the most appropriate technique for researching the viral load as it can quantify the amount of viral nucleic acid present in patient samples.

Question 52

In a study on severe toxicity caused by chemotherapy, what PCR technique would be used to understand the underlying mechanisms?

Answer 52

To understand severe toxicity caused by chemotherapy, RT-qPCR or qPCR could be employed to assess gene expression changes or identify genetic variations.

Question 53

What PCR technique would a neuroscientist use to quantify mRNA levels of neurotransmitter-related genes affected by a cognitive-enhancing drug?

Answer 53

A neuroscientist would use RT-qPCR to quantify mRNA levels of neurotransmitter-related genes, which helps in determining gene expression changes in response to a cognitive-enhancing drug.