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MIC 206 > MICRO Module 6 > Flashcards

MICRO Module 6 Flashcards

(103 cards)

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1
Q

What is PCR?

A

PCR is an enzyme-driven amplification process that can amplify short regions of DNA in vitro.

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2
Q

What does PCR rely on?

A

PCR relies on the use of specific primers that hybridize specifically to target sequences in the DNA sample.

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3
Q

What enzyme is used in PCR?

A

PCR uses a thermostable DNA polymerase enzyme to copy the target DNA.

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4
Q

How is DNA amplified in PCR?

A

Through several repeated cycles of PCR, the target DNA is amplified exponentially.

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5
Q

What are PCR-based diagnostic assays used for?

A

PCR-based diagnostic assays are commonly used to detect pathogens in hospitals.

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6
Q

Which pathogens are routinely detected using PCR?

A

Pathogens like Mycobacterium tuberculosis, Chlamydia trachomatis, and HBV are routinely detected using PCR-based assays.

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7
Q

Why has PCR gained popularity in infectious diseases?

A

The ease of conducting a PCR over conventional cumbersome diagnosis has caused PCR to gain popularity.

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8
Q

What is RT-PCR?

A

RT-PCR is another amplification assay used to detect and quantitate RNA-based genomes like that of SARS-CoV-2.

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9
Q

What are the two steps in RT-PCR?

A

The two steps in RT-PCR are: (1) extraction of RNA from patient specimens, (2) reverse transcription and PCR amplification.

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10
Q

What primers are used in RT-PCR for COVID-19?

A

Primers specific to SARS-CoV-2 are used in RT-PCR.

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11
Q

What regions of the virus are targeted in RT-PCR for SARS-CoV-2?

A

The assay targets regions of the virus nucleocapsid gene (N1 & N3) of SARS-CoV-2.

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12
Q

What is the significance of detecting viral RNA?

A

Detection of viral RNA aids in diagnosis and provides epidemiological and surveillance information.

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13
Q

What is the overall learning objective of the PCR & RT-PCR protocol?

A

Describe how bacteria acquire new genes from their environment by transformation and how the acquisition of novel genes allows them to adapt to their environment.

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14
Q

What is transformation in the context of bacteria?

A

Transformation is the process by which bacteria can obtain genes from other bacterial cells.

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15
Q

Why is transformation important in genetic engineering?

A

Transformation allows for the introduction of new genetic material into bacteria, which is crucial for genetic engineering applications.

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16
Q

What are plasmids?

A

Plasmids are small, circular DNA molecules that can be used in transformation to introduce new genes into bacteria.

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17
Q

What is the purpose of analyzing the results of a transformation experiment?

A

Analyzing results helps determine the success of the transformation and the effectiveness of the introduced genes.

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18
Q

What is the significance of isolating transformed cells from non-transformed cells?

A

Isolating transformed cells is essential to identify which cells have successfully incorporated the new genetic material.

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19
Q

Why are controls important in an experiment?

A

Controls are important to ensure that the results of the experiment are valid and that any observed effects are due to the experimental conditions.

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20
Q

What is PCR?

A

PCR is an in vitro technique that rapidly amplifies DNA to make billions of copies for analysis.

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21
Q

What is required as a substrate for PCR amplification?

A

Only one DNA molecule is required as a substrate for amplification.

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22
Q

What are the three main steps of PCR?

A
  1. Denaturation 2. Annealing 3. Extension
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23
Q

What happens during the denaturation step of PCR?

A

The sample is heated to 95°C, causing the double-stranded DNA to separate into two single strands.

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24
Q

What occurs during the annealing step of PCR?

A

The temperature is lowered to 55°C, allowing primers to bind to the single-stranded template DNA.

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25
What is the role of DNA polymerase in PCR?
DNA polymerase replicates the template strand by attaching complementary deoxyribonucleotides to the end of the primer.
26
How many cycles are typically repeated in PCR?
The steps are repeated 30-40 times.
27
What is RT-PCR?
RT-PCR is a variation of PCR that amplifies RNA by first converting it into complementary DNA.
28
What is the first step of RT-PCR?
The RNA is converted into a complementary DNA copy using the enzyme Reverse Transcriptase.
29
What types of viruses can be detected using RT-PCR?
Viruses like HIV, SARS-CoV, and Influenza, which have RNA as their genomic material.
30
What materials are needed for the PCR experiment described?
pGFP plasmid, master mix, PCR tubes, pipetters, ice bucket, gloves, and a thermocycler.
31
What is the first step in the PCR protocol?
Label the PCR tubes 1 and 2.
32
How much pGFP DNA is added to tube 1?
1 microliter (μL) of pGFP DNA is added to tube 1.
33
What temperature is set for the annealing step in the PCR program?
The annealing temperature is set to 55 degrees Celsius.
34
What is agarose gel electrophoresis?
A technique used to resolve fragments of nucleic acids on a gel-like matrix in the presence of an electric current.
35
How does DNA separate during agarose gel electrophoresis?
DNA is separated based on size, with smaller fragments running farther on the gel than larger ones.
36
What is the purpose of using agarose gel electrophoresis?
To visualize the amplified DNA fragment, often in conjunction with PCR and RT-PCR.
37
What are the learning objectives of agarose gel electrophoresis?
1. Describe the basis of the separation of DNA fragments using agarose gel electrophoresis. 2. Analyze the results of DNA gel electrophoresis and applications. 3. Predict expected results of an experiment. 4. Analyze results in relation to applications like paternity testing and crime scene analysis.
38
What information can be determined using gel electrophoresis?
The presence of DNA in a sample, characteristics such as the number of base pairs, and the amount of DNA.
39
How is agarose gel prepared?
By dissolving agarose in buffer, adding Ethidium Bromide dye, and pouring into a gel tray to solidify.
40
What is the role of the DNA Standard in gel electrophoresis?
It contains a mixture of different DNA molecules of known sizes and amounts, used as a reference to compare the sample DNA.
41
What happens to DNA when subjected to an electric field in gel electrophoresis?
Negatively charged DNA migrates out of the well towards the positive electrode, separating based on size.
42
How are DNA bands visualized in agarose gel electrophoresis?
By binding to Ethidium Bromide dye, which fluoresces under UV light.
43
What does the intensity of a DNA band indicate?
The amount of DNA in that band, with greater intensity indicating more DNA.
44
What applications does DNA electrophoresis have?
Used in molecular biology, medicine, forensic science, DNA fingerprinting, and paternity testing.
45
How is DNA fingerprinting used in paternity testing?
By comparing the DNA patterns of the child, mother, and suspected father to identify matches.
46
What is the application of PCR and DNA gel electrophoresis in diagnostics?
PCR and DNA gel electrophoresis are used to diagnose infections such as Chronic Hepatitis caused by viruses like Hepatitis B (HBV) and Hepatitis C (HCV).
47
What is the principle behind agarose gel electrophoresis?
Agarose gel electrophoresis separates nucleic acids based on their size by applying an electric field.
48
Which patients are infected with HCV, co-infected with HBV and HCV, and have no infection?
Patients are identified based on the presence of positive amplification in the RT-PCR results for HCV and PCR results for HBV.
49
In which direction do DNA molecules move in the gel?
DNA molecules move from the negative electrode (A) to the positive electrode (B) due to their negative charge.
50
What steps were done in Figure 2 to amplify the viral nucleic acid?
Blood samples were subjected to PCR to amplify the HBV gene before resolving the products by agarose gel electrophoresis. This differs from Figure 1, where RT-PCR was used for HCV amplification.
51
What is the purpose of negative and positive controls in this experiment?
Negative and positive controls ensure the reliability of the PCR results by confirming the absence or presence of the target virus.
52
What is PCR?
PCR is an enzyme-driven amplification process that can amplify short regions of DNA in vitro.
53
What is the role of primers in PCR?
Primers hybridize specifically to target sequences in the DNA sample.
54
What enzyme is used in PCR?
A thermostable DNA polymerase enzyme is used to copy the target DNA.
55
How is DNA amplified in PCR?
Through several repeated cycles of PCR, the target DNA is amplified exponentially.
56
What are PCR-based diagnostic assays used for?
They are commonly used to detect pathogens in hospitals.
57
Which pathogens are routinely detected using PCR?
Pathogens like Mycobacterium tuberculosis, Chlamydia trachomatis, and HBV.
58
Why has PCR gained popularity in infectious diseases?
It is easier to conduct than conventional diagnosis, which involves growing organisms under specialized conditions.
59
What is RT-PCR?
RT-PCR is an amplification assay used to detect and quantitate RNA-based genomes like that of SARS-CoV-2.
60
What are the two steps of RT-PCR?
1. Extraction of RNA from patient specimens. 2. Reverse transcription and PCR amplification with specific primers.
61
What regions of the SARS-CoV-2 virus are targeted in RT-PCR?
Regions of the virus nucleocapsid gene (N1 & N3).
62
What is the significance of detecting viral RNA in RT-PCR?
It aids in the diagnosis of illness and provides epidemiological and surveillance information.
63
What is the overall learning objective of the PCR & RT-PCR protocol?
Describe how bacteria acquire new genes from their environment by transformation and how the acquisition of novel genes allows them to adapt to their environment.
64
What are the detailed learning objectives of the PCR & RT-PCR protocol?
1. Describe how bacteria can obtain genes from other bacterial cells by transformation. 2. Discuss the importance of transformation in genetic engineering. 3. Define plasmids and describe their use in transformation. 4. Analyze the results of a transformation experiment. 5. Predict expected results of an experiment. 6. Discuss how to isolate transformed cells from non-transformed cells. 7. Discuss the importance of controls in an experiment.
65
What is PCR?
PCR is an in vitro technique that can rapidly amplify DNA to make billions of copies which can be further analyzed.
66
What is the significance of PCR in various fields?
PCR is used in biological research, diagnosis of infectious disease, and genetic analysis of certain traits.
67
What are the three main steps of PCR?
1. Denaturation 2. Annealing 3. Extension
68
What happens during the denaturation step of PCR?
The sample is heated to 95°C, causing the double-stranded DNA to separate into two single strands.
69
What occurs during the annealing step of PCR?
The temperature is lowered to 55°C, allowing primers to bind to the single-stranded template DNA.
70
What is the purpose of the extension step in PCR?
The temperature is increased to 72°C, and DNA Polymerase replicates the template strand, extending the primers to create new DNA strands.
71
How many cycles are typically repeated in PCR?
The steps are repeated 30-40 times.
72
What is RT-PCR?
RT-PCR is a variation of PCR that amplifies RNA instead of DNA.
73
What are the two steps involved in RT-PCR?
1. Conversion of RNA into complementary DNA using Reverse Transcriptase. 2. Amplification of the complementary DNA through regular PCR.
74
What is the purpose of the PCR protocol in this experiment?
To amplify the GFP gene in the plasmid, pGFP, using primers specific for the GFP gene.
75
What materials are needed for the PCR experiment?
pGFP plasmid, master mix, PCR tubes, pipetters, ice bucket, gloves, thermocycler.
76
What is the first step in the PCR protocol?
Label the PCR tubes 1 and 2.
77
How much pGFP DNA is added to tube 1?
1 microliter (μL) of pGFP DNA is added to tube 1.
78
What temperature is set for the annealing step in the thermocycler?
55 degrees Celsius.
79
What should you consider when comparing PCR and RT-PCR techniques?
Think about the differences in starting templates and the processes involved.
80
What product would you visualize in the PCR of the GFP gene?
The amplified GFP gene product.
81
What is agarose gel electrophoresis?
A technique used to resolve fragments of nucleic acids on a gel-like matrix in the presence of an electric current.
82
How is DNA separated in agarose gel electrophoresis?
DNA is separated based on size, with smaller fragments running farther on the gel than larger ones.
83
What is the purpose of using a DNA standard in gel electrophoresis?
The DNA standard contains a mixture of different DNA molecules of known sizes and amounts, used as a reference to compare the sample DNA with.
84
What is the role of Ethidium Bromide in agarose gel electrophoresis?
Ethidium Bromide is a fluorescent dye that binds to DNA, allowing visualization of DNA bands under UV light.
85
What information can be obtained from gel electrophoresis?
The presence of DNA, characteristics such as the number of base pairs (size), and the amount of DNA in a sample.
86
What happens to DNA when subjected to an electric field in gel electrophoresis?
Negatively charged DNA migrates out of the well towards the positive electrode, separating based on size.
87
What is the significance of band intensity in gel electrophoresis?
The intensity of a DNA band is directly related to the amount of DNA in that band.
88
How is agarose gel prepared for electrophoresis?
Agarose is dissolved in buffer, mixed with Ethidium Bromide, poured into a tray, and allowed to solidify.
89
What applications does DNA gel electrophoresis have?
It is used in molecular biology, medicine, forensic science, DNA fingerprinting, and paternity testing.
90
How is paternity testing conducted using agarose gel electrophoresis?
DNA samples from the child, mother, and suspected father are compared after being resolved on the gel to identify matching patterns.
91
What is the application of PCR and DNA gel electrophoresis in diagnostics?
PCR and DNA gel electrophoresis are used to diagnose infections such as Chronic Hepatitis caused by viruses like Hepatitis B (HBV) and Hepatitis C (HCV).
92
What are the main causes of Chronic Hepatitis?
The top causes of Chronic Hepatitis are Hepatitis B (HBV) and Hepatitis C (HCV).
93
How are Hepatitis B, C, and D transmitted?
Hepatitis B, C, and D are spread by contact with infected blood and contaminated syringes.
94
What was the method used to test for Hepatitis C virus (HCV) in patients?
Blood samples were taken, RNA was extracted, and the samples were amplified by RT-PCR.
95
What does the agarose gel electrophoresis show in the context of HCV testing?
The agarose gel electrophoresis shows the positive amplification of HCV in the tested samples.
96
What was the method used to test for Hepatitis B virus (HBV) in patients?
Blood samples were subjected to PCR to amplify the HBV gene.
97
What does the agarose gel electrophoresis show in the context of HBV testing?
The agarose gel electrophoresis shows the positive amplification of HBV in the tested samples.
98
What is the principle behind agarose gel electrophoresis?
Agarose gel electrophoresis separates nucleic acids based on their size.
99
How can you determine which patients are infected with HCV, HBV, both, or neither?
By analyzing the RT-PCR and PCR results from the gel electrophoresis, you can identify positive amplifications.
100
In which direction do DNA molecules move in the gel?
DNA molecules move from the negative electrode (A) to the positive electrode (B).
101
What are the steps taken to amplify the viral nucleic acid for HBV testing?
Blood samples were subjected to PCR to amplify the HBV gene.
102
How does the amplification process for HBV differ from that for HCV?
HBV amplification uses PCR, while HCV amplification uses RT-PCR.
103
What is the purpose of negative and positive controls in this experiment?
Negative and positive controls ensure the reliability and accuracy of the test results.

MIC 206 (4 decks)

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