Complete the following tasks.

You are fresh recruits to a molecular biology laboratory, and your new boss has
tasked you to study mutations in NRAS, a gene that she suspects is involved in
cancer pathogenesis.

Task A: Polymerase Chain Reaction Master Mix

Your first task is to isolate and amplify the NRAS gene from cDNA extracted from
different samples through PCR. You will need to run a total of 7 PCRs: 3 normal
samples, 3 cancerous samples, and 1 negative control. To make things easier in the
lab, when running multiple reactions, the components are prepared not
individually, but as a master mix—all the components for multiple reactions are
prepared in bulk, except for the template DNA, which is added separately once the
master mix has been distributed into individual tubes.

The table below lists the different components for PCR, the available stock
concentrations of these components, and the needed working concentrations for
the PCR itself. Complete the table by supplying the needed volumes of each
component for a single reaction and for the master mix (the first row has been
filled in as an example).

PCR Stock Working Volume for 1 Volume for 7

Component concentration concentration reaction (μL) reactions (μL)
Buffer for Taq
10X 1X 1.0 7.0
polymerase
Forward
40 uM 1.0 uM 0.25 1.75
primer
Reverse
10 uM 1.0 uM 1.0 7.0
primer
dNTPs 25 mM 1 mM 0.4 2.8
Taq
200 U/ul 1 U/ul 0.05 0.35
polymerase
Sterile
(to fill reaction
distilled
n/a volume to 10 6.3 44.1
deionized
ul)
water
Total 9.0 ul 63.0 ul
1.0 (added separately to each
Template 400 ng/ul 40 ng/ul reaction after master mix is
distributed to different tubes)

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 Total 10.0 70.0

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 Task B [5 points]: Sanger sequencing
Having successfully amplified the NRAS gene from wild-type and cancerous tissue
through PCR, you now sent your PCR product for sequencing. However, the only
available technology to you is classical Sanger sequencing or the dideoxy chain
termination method, explained in Figure 2. Shown below are the sequencing gels
obtained for the sections of the wild-type and mutant NRAS genes where the
mutation can be found.

1. Determine the 5'-to-3' sequence of the wild-type TEMPLATE strand.

Answer: 5’ ACCTGCTCC 3’
2. Using the codon table given below, determine the corresponding wild-type and
mutant amino acid sequences in order to determine the mutation that occurred.
Assume that the sequencing template is the CODING strand for the NRAS gene.
(Use the 3-letter abbreviations for the amino acids.)

Wild-type amino acid sequence: Thr-Cys-Ser

Mutant amino acid sequence: Ile-Cys-Ser
Mutation: The original amino acid _Thr_ was mutated into _Ile_
Bonus : In your PCR master mix, you used Taq polymerase. What species is this
enzyme derived from? Answer: Thermus aquaticus

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