Part of the CFTR gene sequence NOTE - make sure you print or view this page 5\'

Question

Part of the CFTR gene sequence
NOTE - make sure you print or view this page
5' GATGCCGTGCTCCAGGCAC "AATCTCT" TCCAGAACTCGAAACCAACTG
AATGGAGTGATCTCGATCAAGCCGGAGCTTCTCTTAACTTCGATTTCGAC
GTGAAGAACTTACTTTGATTGCCACATTGGCCCAATTGAAGGGTTATAAT
CTCGCTGGGCATTTTGCTCGAATTGCAGTTTAGTTAATTTCGCTTCTTTC
TCAGTAAATAATAACTGTGCATCGAAGGCCAACTGGAGATGGAAGTGGAA
GTCATTAATATCGATATACAAGAAGTACAATACAACAGTAAGTTATTACC
Mutation 1
"AATCTCT"
Deleted bases in
some patients

TATTTTTCTTTACTAATCTTTATGATTTATTTAATGTTATTGGACTTATA
GAAAATACTTAGAATTAAGATTTGATCCATTTTTTTTTTAAATATTTAGG
TTCAAATAAATTAGCCATAGTTATATATTTTTTATGTTTTCATTTACATA
AAACATTAACTGTTATTTTTTTAAATTTAAAGGACTAGCTTTTTCAATTG
TACAGCACACCCTGTGGCTGCGAGTAACGGTTCTCGGGAGTCGTTATTTT
+ 1 CTGAGAAGC "G" CGCAACGTGCACACTGGCAGGCTGAT'rGAAAAl'J'IlrlTCGTT
GCAAATGTTTATGTACAAATATTGAATAAAAAATAAAA ATG CTGGCAG
GAGAACTTGGCAGCCAACTTCTGCGGTCTCCTGGCCAGCCAGGGCTATAA
AGGTGAGTTCGTATTTCGCCAATTTACTCCGAGCCTCATGGATGTCCGTA
Mutation 2
"G" changed to A
in some patients

Mutation 3
"C" changed to T in some patients:

AAAATCGCAGAGAAGGCAAACGAGTGGCGCATTTTGGGC "C" AGGAACAGGA
TGGATCTCTGCTCACGTCCTC G ATATTCGAGTACGCGGACGAGGATCAGC
GCAAGGAGACGTGCATTGGCCACTTTCACGCCACCAAGAAGCAGCTGCGA
CTCCTTTGGACCCTCGACAATTGCCGTGAGAI'CGTCCAGGCAACGATTAA
CAGCAGTGTCACATTGCTGTCCTTCGTGGAGAAAACTGAGGGCAAGCTCT
ATCAGGCCTTTGTCGTGGAGGTAAGGAGCTCCGAAGGTGGCACGGCCACG
CCCCTCAACTCGGAGCCCTCCAACCGCCAGATGATGACGCAGTTCCTGTG
GCGCGTCGAGAGTGCCACGCGCACCTGCTGGCAGGACAAGCTACTGGTGC
TCACCCACGAGGAATGTAGGTGTCATGTATATGTCATTATCCACCTC~
CTCCCATGCTCGTACCTGTTTTTTCTAGCCATCAAGCAGTACAGCTGCGT
GGTCAAGCAGAGCTCCACCACATGCTCGACTGGCGGAGGCGAGGGCAGCG
CCTGGAGGCTAGACACCAGCATACTGACCTACGAAACGCTGGCCAGGAAC
TTTAGCTGGGCCCAGTGGGATCCCGAGTGCCAGGCTCTTTATTACATTCA

Mutation 4
"G" changed to C
in some patients:
CTTGAAGCCGAAGGCCAAGAGCCTCAGTCTGCTGGACGAGAGGGAGGAGG
CTGGCGAGCAGACAACTCCTACTTTAAGCCCCACGCTCTCCGCCTTTCAG
TTTAAC~AGCCAACGGAAACAGTGGTAGGTACTTl'AAGATTAAA
TGACTGTGCGTTAGGTCCTGTTCATTGGTACCCTTTATGAGTTAATTCAA
ACCCACGGACATGCTAAGGGTTAATAAACAATCATATCGCTGTCTC

The above figure shows a sequence of the CFTR gene and mark clearly where your PCR-amplified region of the DNA lies.

Design a genetic test based on a PCR reaction:

Your company has decided to develop a genetic test that will be used to diagnose the genotype of candidate CF patients evaluate their risk of developing the disease. Due to cost consideration, this genetic test will consist of a single PCR-amplification reaction of genomic DNA. The amplified fragment should be around 200 base pairs. Although cellular DNA replication employs RNA primers, PCR reactions commonly use DNA primers, so design your primers to be made of DNA.

According to the data, it's found that the 3rd mutation is the most likely to cause the disease of cystic fibrosis. Design two primers (one forward and one reverse) to be used in the PCR reaction that will amplify a fragment of approximately 200 base pairs that includes the mutation. You should aim to have the mutation approximately in the middle of the mutation. Each primer should be 18 nucleotides long and should be at least 50% GC-rich.

Please write the sequence of your selected primers in the provided space (marking the 5' and the 3' direction of the DNA) and also underline the sequences that you oicked in the genomic fragment that you are given. Also, add the % GC content of your primers in the provided space. Both primers should be written in the 5' to 3' direction.

Forward primer:

---------------------------------------------------------------------------------------------------------------------------------------------------------- % GC --------

Reverse primer:

--------------------------------------------------------------------------------------------------------------------------------------------------------- % GC ---------

Explanation / Answer

5' CCCTTAGCATGTCCGTGG 3'

Mutation 1 forward primer 5’ GATGCCGTGCTCCAGGCA 3’ 66% Mutation 1 reverse primer 5’ GGCCTTCGATGCACAGTT 3' 55 % Mutation 2 forward primer 5’ GCAGCACACCCTGTGGCT 3' 66 % Mutation 2 reverse primer 5’ GCCCTGGCTGGCCAGGAG 3' 77 % Mutation 3 forward primer 5’ CGCAGAGAAGGCAAACGA 3' 55 % Mutation 3 reverse primer 5’ GCCTGGACGTCTCACGGC 3' 72 % Mutation 4 forward primer 5’ AGCCGAAGGCCAAGAGCC 3' 66 % Mutation 4 reverse primer

5' CCCTTAGCATGTCCGTGG 3'

61 %

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