Q4.

How would you classify the patient's sequence

variant at the protein-coding level? (1 mark)

Q2. In the week three applied activities, you performed a pairwise sequence alignment between the patient’s DNA sequencing results and the MFN2 reference sequence (NG_007945.1). Upload a screenshot of your pairwise sequence alignment output from Clustal Omega to the file upload box.

A) PCR RESULTS UPLOAD:

 Upload the

agarose gel image of your PCR results to the file upload box. Record which gel lanes correspond to your experimental reaction, positive control reaction and negative control reaction in the provided table.

B) NEGATIVE CONTROL REACTION

The expected outcome for the negative control reaction is no

visible DNA bands.

i)

Did you observe any visible bands in your negative

control? (YES/NO) (1 mark)

If NO, proceed to the next question (ii). 

If YES, explain whether or not you can have confidence in the

results obtained for the positive control reaction and experimental reaction. In

your answer, consider what the size and appearance of the observed band(s) may

indicate.

C) POSITIVE CONTROL REACTION

The expected outcome for the positive control reaction is a

single DNA band at 187 bp.

ii) Did your positive control produce a band of the

expected size? (YES/NO) (1 mark)

If YES, proceed to the next question (iii). 

If NO, suggest one or more sources of error or procedural

issues that could have resulted in failed amplification of the target sequence.

iii) Did your positive control produce an additional band(s)

at unexpected sizes? (YES/NO) (1 mark)

If NO, proceed to the next question (iv). 

If YES, explain whether or not these bands represent

non-specific amplification. If they do represent non-specific amplification,

suggest one or more adjustments to the reaction mix or thermal cycling

conditions that could improve the specificity of the PCR.

D) EXPERIMENTAL REACTION

The expected outcome for the experimental reaction is a

single DNA band at 187 bp.

iv) Did your experimental reaction produce a band of the

expected size? (YES/NO) (1 mark)

If YES, proceed to the next question (v). 

If NO, suggest one or more sources of error or procedural

issues that could have resulted in failed amplification of the target sequence.

v

) Did your experimental reaction produce an additional

band(s) at unexpected sizes? (YES/NO) (1 mark)

If NO, proceed to the next quiz question. 

If YES, explain whether or not these bands represent

non-specific amplification. If they do represent non-specific amplification,

suggest one or more adjustments to the reaction mix or thermal cycling

conditions that could improve the specificity of the PCR.

Which of the following reagents is suspected of causing genetic defects? (select all that apply)

Guanidine hydrochloride (CW No. 8793256) is a component of the binding buffer. Which of the following hazard statements are listed in the mini SDS for this reagent? (select all that apply)

You will set-up three separate PCR reactions in this laboratory class: experimental reaction, positive control reaction, negative control reaction. 

Which of the following reaction components are included in the negative control reaction? (select all that apply)

The PCR mastermix will be prepared in a sterile microtube that must be handled using aseptic technique. Why is this important?

Use the dilution equation (C1V1 = C2V2) to calculate the volume (µL) of 2mM dNTPs that is needed to prepare 100µL of PCR mastermix with a final dNTP concentration of 200µM.

DNA concentration and purity can be assessed by measuring the absorbance of ultraviolet (UV) light at three different wavelengths: 230nm, 260nm and 280nm. 

Which molecule absorbs UV light most strongly at 260nm?

A silica column-based method will be used to purify the patient's DNA. In the presence of ethanol and high salt, which molecule binds strongly to silica?