To complete the assignment, answer questions 1-12.

To complete the assignment, answer questions 1-12.

Case Study:

Tay-Sachs disease is an autosomal recessive disorder affecting the central nervous system. Homozygous recessive individuals with the disorder have progressive degeneration of the central nervous system, which is usually fatal. A couple have been referred to a medical clinic for genetic counselling in regards to their present pregnancy. Both the couples family have a history of Tay-Sachs disease. After consultation with the couple, a pedigree of the family has been constructed (Figure 1). The couple are individuals VI2 and VI3 on the pedigree.

Figure 1: Pedigree of a family with individuals with Tay-Sachs disease.

Question 1

What is the probability of the couple (individuals VI2 and VI3 on the pedigree) having a child with Tay-Sachs disease. Show calculations.

The couple decide to have an amniocentesisfor prenatal genetic diagnosis totestfor the presence of a mutation in theTay-Sachs gene in their unborn child. Tay-Sachs disease is caused by the absence of an enzyme called hexosaminidase A which is encoded by the gene hexA. Without hexA, a fatty substance builds up on the nerve cells in the body. A region of DNA encompassing the hexA gene has been amplified using PCR from the amniocentesis and cloned into the pCR2.1-TOPO vector (Figure 2).

Figure 2. Map of the vector pCR 2.1-TOPO.

The recombinant plasmid has been digested with a variety of restriction enzymes and these digests have been analysed by gel electrophoresis. The gel photo is shown in Figure 3. Use the gel photo of the restriction fragments to answer the following questions (hint: use the second gel photo of the DNA ladder (Figure 4) to first identify the sizes of the ladder bands on the first gel (Figure 3)):

Figure 3. Gel photo of the restriction enzyme digests of the recombinant plasmid generated by cloning the hexA gene PCR product from the amniocentesis of the unborn child into the pCR2.1-TOPO vector.

Figure 4. Gel photo of the DNA size ladder showing the relative size of each band in bps.

Question 2

What is the total size of the recombinant plasmid and what is the size of the PCR insert? Explain how you calculated these.

Question 3

One researcher in the team wants to digest the recombinant plasmid with the restriction enzyme EcoRI to subclone the entire PCR insert into a different vector. Explain why this would/would not be an appropriate choice of enzyme to use for this purpose.

Question 4

Which combination of two restriction enzymes can be used to cut out the entire PCR insert (without cutting within the insert)?

The M13 Reverse primer and M13 Forward (-20) primer were used to sequence (Sanger sequencing) the PCR product cloned into pCR2.1-TOPO. A segment of the sequencing chromatogram is shown in Figure 5.

Figure 5. A. Region of the sequence chromatogram generated from the recombinant plasmid. B. corresponding sequence of a region of the hexA gene from a wildtype control. The reading frame (codons) are indicated by the black lines below the sequence.

Question 5

What type of DNA mutation is present in the fetus? Does this cause a mutation in the HexA protein and if so, what is this? (hint: use the genetic code to translate the DNA sequence)

Question 6

Can you identify if the fetus is homozygous or heterozygous for a mutation in hexA? Explain how you determined this and would you expect the fetus to have Tay-Sachs disease?

The researchers want to confirm that the mutated hexA gene can still produce a protein product. They aim to clone the hexA gene into another vector containing the gene GFP and then observe if a GFP::HexA fusion protein is produced. A map of the GFP vector pcDNA3.1 NT-GFP-TOPO is shown in Figure 6. Analyze the vector map and answer the following questions.

Figure 6. Map of the vector pcDNA3.1 NT-GFP. Genes are indicated by arrows, plasmid features by boxes. Recognition sites for restriction sites and their relative locations in the plasmid are indicated (unique sites shown in bold).

Question 7

Which restriction enzyme/s will cut the plasmid within the open reading frame (ORF) of the GFP gene?

Question 8

Which combination of two restriction enzymes could be used to cut out the smallest possible fragment of the plasmid that contains the CMV enhancer and GFP but not the bGH poly(A) signal? What is the size of this fragment?

Question 9

The combination of BsrG1 and KpnI cannot be used to cut out the entire (intact) GFP gene from the vector. Why?

Question 10

What is the expected sizes of the restriction fragment/s generated from a double digestion of this plasmid with both HindIII and PfoI?

Question 11

Describe how you would sub-clone the KpnI/NotI fragment from the recombinant clone into the vector pcDNA3.1 NT-GFP (Figure 6).

* Include the steps on generating the recombinant molecule, amplifying the recombinant plasmid in bacteria and how you would select for the recombinant clone in E. coli. Include explanations/justification of why you would perform each step.

Note: You do not have the sequence or any primers so you cannot use PCR.

Question 12

Use the restriction enzyme recognition sites listed below to explain if it is possible to sub-clone the SpeI/NotI fragment from the recombinant clone into the vector pcDNA3.1 NT-GFP digested with XbaI/NotI.

SpeI

XbaI

NotI

BIOL2262 Assignment 2 Rubric

BIOL2262 Assignment 2 Rubric

Criteria Ratings Pts

This criterion is linked to a learning outcomeCriteria 1

Predicting experimental results using a human pedigree (CLO1) demonstrates evidence of problem solving and critical thinking (CLO1) capability which can be applied in real world examples 20Pts

Outstanding

There is strong and convincing evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The probability in question 1 was correctly calculated using the product rule and all calculations were shown. 15Pts

Advanced

There is strong evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The probability calculations in question 1 either contained one error or omission in the calculations. 12.5Pts

Developed

There is reasonable evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The probability calculations in question 1 either contained two errors or omissions in the calculations. 10Pts

Competent

There is limited evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The probability calculations in question 1 contained many errors or omissions but a basic understanding of the principles of inheritance was demonstrated. 0Pts

Not Satisfactory

There is insufficient evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome.

20pts

This criterion is linked to a learning outcomeCriteria 2

Interpreting experimental data (CLO1) demonstrates a) an understanding of recombinant DNA technology and b) an ability to analyse and interpret experimental results of gel electrophoresis of restriction digests of a recombinant plasmid in a scientifically meaningful way (CLO2). 30Pts

Outstanding

The interpretation of the experimental data shows an advanced and thorough understanding of recombinant DNA technology and a very high level and significant capability to analyse and interpret experimental results which are unambiguous and strong in validity and have scientific accuracy and meaning. The sizes in question 2 were calculated correctly (showing size units), the answer to question 3 was correct with a reasonable explanation and question 4 was answered correctly. 22.5Pts

Advanced

The interpretation of the experimental data shows a sound and reasonably comprehensive understanding of recombinant DNA technology and a high level ability to analyse and interpret experimental results which are strong in validity and scientific accuracy and meaning. The answers to questions 2-4 contained an error or omission. 18.75Pts

Developed

The interpretation of the experimental data shows a good understanding of recombinant DNA technology and an ability to analyse and interpret experimental results which are clear and scientifically useful. The answers to questions 2-4 contained 2 errors or omissions. 15Pts

Competent

The interpretation of the experimental data shows an adequate understanding of recombinant DNA technology and a limited capability in interpreting and analysing experimental results which may have some scientific application. The answers to questions 2-4 contained 3 errors or omissions. 0Pts

Not Satisfactory

The interpretation of the experimental data shows an inadequate understanding of recombinant DNA technology and an unsatisfactory capability in interpreting and analysing experimental results. No scientific conclusions can be drawn.

30pts

This criterion is linked to a learning outcomeCriteria 3

Analysis of the chromatogram sequence file demonstrated an ability to manage tasks (CLO1) commonly performed in a molecular biology laboratory. 30Pts

Outstanding

Analysis of the DNA sequence was undertaken demonstrating exemplary skills and ability to execute and manage tasks commonly performed in a lab environment. Answers to questions 5 and 6 correctly identified the DNA mutation and consequence in the predicted protein, if homozygous or heterozygous and if they had Tay-Sachs disease and included a reasonable explanation. 22.5Pts

Advanced

Analysis of the DNA sequence was undertaken demonstrating high-level skills in executing and managing tasks commonly performed in a lab environment. Answers to questions 5 and 6 contained 1 error or omission. 18.75Pts

Developed

Analysis of the DNA sequence was undertaken demonstrating good skills in executing and managing tasks commonly performed in a lab environment. Answers to questions 5 and 6 contained 2 errors or omissions. 15Pts

Competent

Analysis of the DNA sequence was undertaken demonstrating a beginners level capability in the execution and management of tasks commonly performed in a lab environment. Answers to questions 5 and 6 contained 3 errors or omissions. 0Pts

Not Satisfactory

Analysis of the DNA sequence was unsatisfactory demonstrating an unsatisfactory ability to execute and manage tasks commonly performed in a lab environment.

30pts

This criterion is linked to a learning outcomeCriteria 4

Predicting experimental results using a restriction map (CLO1) demonstrates evidence of problem solving and critical thinking (CLO1) capability which can be applied in real world lab environments 30Pts

Outstanding

There is strong and convincing evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 7 and 8 were correct (must include units of size measurement) 22.5Pts

Advanced

There is strong evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 7 and 8 contained 1 error or omission. 18.75Pts

Developed

There is reasonable evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 7 and 8 contained 2 errors or omissions. 15Pts

Competent

There is limited evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 7 and 8 contained 3 errors or omissions 0Pts

Not Satisfactory

There is insufficient evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments.

30pts

This criterion is linked to a learning outcomeCriteria 5

Predicting experimental results using a restriction map (CLO1) demonstrates evidence of problem solving and critical thinking (CLO1) capability which can be applied in real world lab environments 20Pts

Outstanding

There is strong and convincing evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 9 and 10 were correct (must include units of size measurement) 15Pts

Advanced

There is strong evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 9 and 10 contained 1 error or omission. 12.5Pts

Developed

There is reasonable evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 9 and 10 contained 2 errors or omissions. 10Pts

Competent

There is limited evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments. Answers to questions 9 and 10 contained 3 errors or omissions. 0Pts

Not Satisfactory

There is insufficient evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome and which are commonly assigned when working in biological laboratory environments.

20pts

This criterion is linked to a learning outcomeCriteria 6

Planning a sub-cloning experimental strategy (CLO1) demonstrates a) an understanding of recombinant DNA technology and b) understanding of molecular cloning and communicating this in a scientifically meaningful way (CLO2). 60Pts

Outstanding

Designing a sub-cloning experimental strategy shows an advanced and thorough understanding of recombinant DNA technology and a very high level and significant capability to plan experiments which are unambiguous and strong in validity and have scientific accuracy and meaning. In answer to question 11, included 4 key steps of the sub-cloning strategy with clear explanations/justifications. 45Pts

Advanced

Designing a sub-cloning experimental strategy shows a sound and reasonably comprehensive understanding of recombinant DNA technology and a high level ability to plan experiments which are strong in validity and scientific accuracy and meaning. Included 4 key steps of the sub-cloning strategy with a minor error or omission in the explanations. 37.5Pts

Developed

Designing a sub-cloning experimental strategy shows a good understanding of recombinant DNA technology and an ability to plan experiments which are clear and scientifically useful. Included at least 3 key steps of the sub-cloning strategy and/or minor errors or omissions in the explanations. 30Pts

Competent

Designing a sub-cloning experimental strategy shows an adequate understanding of recombinant DNA technology and a limited capability in planning experiments which may have some scientific application. Included at least 2 key steps of the sub-cloning strategy and/or errors or omissions in the explanations. 0Pts

Not Satisfactory

Designing a sub-cloning experimental strategy shows an inadequate understanding of recombinant DNA technology and an unsatisfactory capability in planning experiments. No scientific conclusions can be drawn.

60pts

This criterion is linked to a learning outcomeCriteria 7

Analysis of the recognition sites for restriction enzymes demonstrates an understanding of recombinant DNA technology (CLO1). 10Pts

Outstanding

There is strong and convincing evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The answer to question 12 shows an advanced and thorough understanding of recombinant DNA technology. 7.5Pts

Advanced

There is strong evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The answer to question 12 shows a sound and reasonably comprehensive understanding of recombinant DNA technology. Contains a minor error. 6.25Pts

Developed

There is reasonable evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The answer to question 12 shows a good understanding of recombinant DNA technology. Contains a major error or omission. 5Pts

Competent

There is limited evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome. The answer to question 12 shows an adequate understanding of recombinant DNA technology. Contains two major errors or omissions. 0Pts

Not Satisfactory

There is insufficient evidence of an ability to think critically and problem solve when executing multifaceted scientific tasks which have a clear outcome.

10pts

Total points:200