BIOLO-In practical 3 you have learned how HPV can

In practical 3 you have learned how HPV can be detected from formalin-fixed paraffin embedded blocks, using primers GP5+/GP6+. Answer the following questions based on the agarose gel image in figure 1. Looking at figure 1, what can you say about the HPV status of this patient? Make sure to explain in your answer how you reached your conclusion. In your answer, make sure you comment on the results showed in all experimental lanes.

Answer

The HPV status of the patient cannot be determined from the provided information.

Solution

a

Marker lane (Lane 1): The DNA ladder provides a reference for estimating the size of DNA fragments in other lanes

b

Empty lane (Lane 2): This lane is intentionally left empty and does not provide information about the HPV status

c

Positive control (Lane 3): The presence of a specific band pattern would confirm that the experiment is working correctly. However, the band pattern is not described in the provided information

d

Sample lane (Lane 4): This lane contains the DNA sample of interest. To determine HPV status, we would expect to see a band corresponding to the size of the amplified HPV DNA fragment using GP5+/GP6+ primers. The presence or absence of such a band is not described in the provided information

e

Negative control (Lane 5): This lane should not contain any DNA bands if the experiment is free from contamination. The presence or absence of bands in this lane is not described in the provided information

f

Unlabeled lane (Lane 6): Without a description or purpose, this lane cannot provide information about the HPV status

Key Concept

Determining HPV status using agarose gel electrophoresis requires analysis of specific band patterns corresponding to the amplified HPV DNA fragments.

Explanation

To conclude on the HPV status of a patient, the gel should show a band of the expected size in the sample lane, which would indicate the presence of HPV DNA when using specific primers. The absence of such a band would suggest a negative result. However, without information on the band patterns in the sample and control lanes, no conclusion can be drawn.

In practical 3 you have learned how HPV can be detected from formalin-fixed paraffin embedded blocks, using primers GP5+/GP6+. Answer the following questions based on the agarose gel image in figure 1. a) Looking at figure 1, what can you say about the HPV status of this patient? Make sure to explain in your answer how you reached your conclusion. In your answer, make sure you comment on the results showed in all experimental lanes. b) Based on the results shown in figure 1, what can you tell your patient about the risk of developing cancer? Explain the reasons behind your answers. c) Still considering results shown in figure 1, would you recommend this patient to get an HPV vaccination? Explain the reasons behind the answers. d) Based on what you have learned and the results of figure 1, what is the risk of this patient to develop breast cancer? Explain the reasons behind your answers. e) Would you recommend this patient to undergo colposcopy? Explain the reasons behind your answer. f) Assuming you had used a TaqMan probe in this experiment, would the result you obtain different? Explain the reasons behind your answer.

a Answer

The HPV status of the patient is positive for HPV.

Solution

a

The presence of a specific band in the sample lane (Lane 4) at the same position as the positive control (Lane 3) indicates the presence of HPV DNA in the patient's sample. The band labeled "200 bp" corresponds to the expected size of the amplified HPV DNA fragment using primers GP5+/GP6+

Key Concept

Detection of HPV DNA

Explanation

The presence of a band in the sample lane at the same position as the positive control suggests the patient's sample contains HPV DNA, indicating a positive HPV status.

b Answer

The presence of HPV increases the risk of developing certain types of cancer, but additional information is needed to assess the specific cancer risk for this patient.

Solution

b

HPV is associated with an increased risk of developing cervical cancer and other anogenital cancers. The detection of HPV DNA in the patient's sample suggests an increased risk, but the type of HPV and other risk factors must be considered to determine the specific cancer risk

Key Concept

HPV and Cancer Risk

Explanation

HPV infection is a risk factor for certain cancers, but the exact risk depends on the HPV type and other individual risk factors.

c Answer

HPV vaccination is recommended to prevent infection with high-risk HPV types that can lead to cancer.

Solution

c

Since the patient has tested positive for HPV, vaccination could still be beneficial to protect against other high-risk HPV types not detected in the current test

Key Concept

HPV Vaccination

Explanation

Vaccination can protect against multiple high-risk HPV types, potentially reducing the risk of future HPV-related cancers.

d Answer

There is no direct link between HPV and breast cancer; the risk of breast cancer cannot be determined from HPV status alone.

Solution

d

HPV is not typically associated with breast cancer. The risk of breast cancer would be assessed based on other risk factors such as family history, genetic predisposition, and lifestyle factors

Key Concept

HPV and Breast Cancer

Explanation

HPV is primarily linked to cervical and other anogenital cancers, not breast cancer. Breast cancer risk assessment requires different criteria.

e Answer

Colposcopy may be recommended if the patient has abnormal cervical screening results, but additional information is needed to make this recommendation.

Solution

e

A positive HPV test is one criterion for recommending colposcopy, but the decision also depends on cervical cytology results and other clinical factors

Key Concept

Colposcopy Recommendation

Explanation

Colposcopy is considered when there are signs of potential cervical abnormalities, which may be indicated by a positive HPV test in conjunction with abnormal cervical cytology.

f Answer

The use of a TaqMan probe would not change the HPV status result but could provide additional quantitative information.

Solution

f

TaqMan probes are used in real-time PCR to provide quantitative data on DNA amplification. While the presence or absence of HPV DNA would remain the same, the use of a TaqMan probe could give a quantitative measure of viral load

Key Concept

TaqMan Probe in PCR

Explanation

TaqMan probes allow for real-time monitoring and quantification of PCR products but do not change the qualitative result of the presence or absence of the target DNA.

In practical 3 you have learned how HPV can be detected from formalin-fixed paraffin embedded blocks, using primers GP5+/GP6+. Answer the following questions based on the agarose gel image in figure 1. a) Looking at figure 1, what can you say about the HPV status of this patient? Make sure to explain in your answer how you reached your conclusion. In your answer, make sure you comment on the results showed in all experimental lanes. b) Based on the results shown in figure 1, what can you tell your patient about the risk of developing cancer? Explain the reasons behind your answers. c) Still considering results shown in figure 1, would you recommend this patient to get an HPV vaccination? Explain the reasons behind the answers. d) Based on what you have learned and the results of figure 1, what is the risk of this patient to develop breast cancer? Explain the reasons behind your answers. e) Would you recommend this patient to undergo colposcopy? Explain the reasons behind your answer. f) Assuming you had used a TaqMan probe in this experiment, would the result you obtain different? Explain the reasons behind your answer.

a Answer

The patient is likely positive for HPV.

Solution

Key Concept

HPV detection using agarose gel electrophoresis

Explanation

The presence of a specific band in the sample lane that matches the positive control and the absence of bands in the negative control suggest the patient's sample contains HPV DNA.

b Answer

The patient may have an increased risk of developing cancer associated with HPV.

Solution

Key Concept

HPV and cancer risk

Explanation

HPV infection is a known risk factor for the development of certain cancers, and the detection of HPV DNA in the patient's sample indicates an increased risk.

c Answer

HPV vaccination is recommended for individuals who have not been exposed to HPV, but it may not be as beneficial for those already infected.

Solution

Key Concept

HPV vaccination and existing infection

Explanation

The vaccine is most effective in preventing HPV infections before exposure, but it may still offer protection against other strains for a patient already infected with one strain of HPV.

d Answer

The results do not directly indicate an increased risk of breast cancer.

Solution

Key Concept

HPV and breast cancer risk

Explanation

While HPV is a risk factor for several cancers, there is no established direct link between HPV and an increased risk of breast cancer.

e Answer

A colposcopy may be recommended to further assess the extent of HPV infection and potential cervical abnormalities.

Solution

Key Concept

Colposcopy following HPV detection

Explanation

Colposcopy is a diagnostic tool used to examine the cervix for abnormalities after an HPV infection has been detected, which can help in managing the patient's health.

f Answer

The use of a TaqMan probe would not change the HPV status result but could provide a more specific and quantitative assessment.

Solution

Key Concept

TaqMan probe in PCR

Explanation

The TaqMan probe allows for real-time monitoring and quantification of DNA amplification, which can provide more detailed information about the HPV infection.

In practical 3 you have learned how HPV can be detected from formalin-fixed paraffin embedded blocks, using primers GP5+/GP6+. Answer the following questions based on the agarose gel image in figure 1. a) Looking at figure 1, what can you say about the HPV status of this patient? Make sure to explain in your answer how you reached your conclusion. In your answer, make sure you comment on the results showed in all experimental lanes.

Answer

The patient is likely positive for HPV.

Solution

a

Lane 1 (Marker): The DNA ladder provides size references. The 200 bp band is clearly visible, which is important for comparing the size of bands in other lanes

b

Lane 2 (Empty lane): This lane serves as a control or spacer and does not affect the interpretation of the results

c

Lane 3 (Positive control): The presence of a specific band pattern confirms that the experiment is working correctly. The band in this lane should match the band in the sample lane if HPV DNA is present

d

Lane 4 (Sample): The presence of a band that corresponds to the size of the expected HPV DNA fragment (as indicated by the positive control) suggests that the patient's sample contains HPV DNA

e

Lane 5 (Negative control): Ideally, this lane should not show any bands. The presence of a band here indicates possible contamination, which could affect the interpretation of the results

f

Lane 6: Without a description, it is not possible to interpret the results of this lane

Key Concept

Interpreting agarose gel electrophoresis results for HPV detection

Explanation

The presence of a band in the sample lane that matches the positive control indicates HPV DNA is likely present in the patient's sample. The negative control should not show any bands; if it does, it suggests contamination.

In practical 3 you have learned how HPV can be detected from formalin-fixed paraffin embedded blocks, using primers GP5+/GP6+. Answer the following questions based on the agarose gel image in figure 1. a) Looking at figure 1, what can you say about the HPV status of this patient? Make sure to explain in your answer how you reached your conclusion. In your answer, make sure you comment on the results showed in all experimental lanes.

Answer

The patient is likely positive for HPV.

Solution

a

Analysis of Lane 4: The presence of a band in the sample lane that corresponds to the 200 bp marker indicates the presence of HPV DNA, as this is the expected size for the amplification product of the GP5+/GP6+ primers

b

Comparison with Controls: The positive control in Lane 3 shows a band at the same position, confirming the test's ability to detect HPV DNA. The negative control in Lane 5 lacks a band, indicating that the reagents are not contaminated

c

Conclusion: Since the band in the sample lane matches the expected size and the controls behave as expected, the patient's sample is likely positive for HPV

Key Concept

Gel electrophoresis analysis for HPV detection

Explanation

The presence of a DNA band at the expected size in the sample lane, along with appropriate positive and negative controls, indicates HPV positivity.

Answer the following questions based on the agarose gel image in figure 1. a) Looking at figure 1, what can you say about the HPV status of this patient? Make sure to explain in your answer how you reached your conclusion. In your answer, make sure you comment on the results showed in all experimental lanes.

Answer

The patient is likely positive for HPV.

Solution

a

Marker lane: The DNA ladder in lane 1 provides reference points for estimating the size of DNA fragments in other lanes

b

Empty lane: Lane 2 serves as a control to check for contamination. The absence of bands indicates no contamination

c

Positive control: Lane 3 shows a band at the expected size, confirming that the experiment conditions were suitable for DNA amplification

d

Sample lane: Lane 4 shows a band at the same position as the positive control, suggesting the presence of HPV DNA in the patient's sample

e

Negative control: Lane 5 shows no band, indicating that the reagents and the experiment are free from contamination

Key Concept

Interpreting agarose gel electrophoresis results for HPV status

Explanation

The presence of a DNA band in the sample lane that corresponds to the size of the positive control indicates that the patient's sample contains HPV DNA, leading to the conclusion that the patient is likely positive for HPV. The absence of bands in the negative control and empty lane confirms the validity of the experiment.

is there a band in lane 3, if there is, how does it means? why there is a band at the bottom of lane 5 and how does it means?

1 Answer

Presence of a band in lane 3 indicates a positive result for HPV DNA.

Solution

a

Positive control: The band in lane 3 confirms that the assay is working correctly and is capable of detecting HPV DNA if present

b

Interpretation: Since the positive control shows a band, it means that the experimental conditions were set correctly and the reagents were functioning as expected

Key Concept

Positive control in gel electrophoresis

Explanation

The presence of a band in the positive control lane is crucial for validating the experiment. It shows that the assay can detect the target DNA, in this case, HPV DNA.

2 Answer

A band at the bottom of lane 5 suggests possible contamination or non-specific amplification.

Solution

a

Negative control: The band at the bottom of lane 5 is unexpected and indicates a problem, as the negative control should not contain any target DNA

b

Possible causes: This could be due to contamination of the negative control with DNA or primer-dimer formation during PCR, leading to non-specific amplification

Key Concept

Negative control in gel electrophoresis

Explanation

The negative control should not show any bands. A band at the bottom of the negative control lane suggests contamination or non-specific amplification, which could compromise the validity of the experiment.

what can we know from figure 1

Answer

From Figure 1, we can determine the HPV status of the patient by analyzing the DNA bands in Lane 4 (Sample) and comparing them to the controls and the DNA ladder.

Solution

a

Lane 1 (Marker): The DNA ladder provides a reference for estimating the size of DNA fragments in other lanes

b

Lane 3 (Positive control): The presence of a band at the 200 bp position indicates the positive control is working and the experiment is valid

c

Lane 4 (Sample): The presence or absence of a band at the 200 bp position, as indicated by the blue arrow, will reveal the HPV status of the patient. If a band is present at this position, it suggests the patient's sample contains HPV DNA of the expected size

d

Lane 5 (Negative control): The absence of bands in this lane confirms that there is no contamination or false positive in the experiment

Key Concept

Interpreting agarose gel electrophoresis results to determine the presence of specific DNA fragments.

Explanation

By comparing the sample lane to the positive control and the DNA ladder, we can conclude whether the patient's sample contains HPV DNA. The negative control ensures the specificity of the test.

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