Biology

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PCR Primer Design Assignment

Instructions: Refer to the DNA sequence on the following page and the information provided to answer the questions. In a recent study, it was shown that patients with an inherited cancer syndrome have mutations, in the form of insertions (added base pairs) and deletions (deleted base pairs) in the gene for which the sequence is shown on the next page. The area highlighted by the red box is the region in which these mutations are found. Design a set of PCR primers to amplify this region of the gene. These primers and PCR products need to follow the guidelines for making good primers as discussed in class and as outlined on the summary document provided.

1. Design a forward and a reverse primer to amplify the region indicated by the red box on the gene sequence on the following page. (6 pts)

PLEASE NOTE: when ordering primers, sequences are ALWAYS written in the 5’ to 3’ direction. Keep this in mind when writing your sequences for these primers!!

Forward Primer start and end (base pair number in sequence):

Base pair start number: Base pair end number: Primer length:

Forward Primer Sequence, written as 5’ 3’:

5’-

Reverse Primer start and end (base pair number in sequence):

Base pair start number: Base pair end number: Primer length:

Reverse Primer Sequence, written as 5’ 3’:

5’-

2. Calculate the PCR amplicon (product) size for the set of primers you designed. (3 pts)

Amplicon size (bp)

3. Calculate the Tm for each primer. SHOW your work within the right text box. (3 pts)

Primer Tm (C) Show work!! Provide formula that you are using in the first box below. Then in each box below that, write out the information that would go into this equation. Credit will not be given without work shown.
Provide formula to be used here:
Forward primer Tm:
Reverse primer Tm:

4. Based on the information above and the gene sequence, design a PCR cycle for this assay. (3 pts)

Step: Temperature (C) Time – INDICATE time & units [minutes (min) or seconds (sec)]
Denaturation 94
Annealing
Elongation 72

DNA Sequence for PCR Assignment

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