PCR screening for the L13F polymorphism: Computer Exercise

Aim 1. To perform precise DNA sequence analysis of the COL1A2 gene.

Aim 2. To investigate how mutagenic primers, PCR and restriction enzyme digestion can be used as tools to diagnose the presence of disease-causing mutations.

Diagnostic Method

A PCR-based procedure is used to screen genomic DNA for the L13F COL1A2 polymorphism. An overview of the method is provided below. Understanding of this section is crucial before proceeding to the questions. Take time to read all below information carefully.

PCR primers complementary to the COL1A2 sequence are first designed. Three nucleotide substitutions are intentionally introduced in one of the primers (the antisense primer L13F2). As a result of the nucleotide substitutions introduced by this mutagenic primer, amplification of the wild type COL1A2 gene produces a PCR product with a sequence slightly different to that of the wild-type gene. Key to this diagnostic procedure is that this slightly altered PCR product can now be digested by the restriction enzyme EcoNI.

A PCR product using the same primer pair, but the mutant COL1A2 gene containing the L13F polymorphism as a template cannot be digested by EcoNI. The resultant size difference between an EcoNI digested PCR product from a wild-type vs a mutant gene can be resolved by agarose gel electrophoresis.

The primers used are:

Sense primer L13F1: 5'- CCGCCAGGTGATACCTCCGCC - 3'

Antisense primer L13F2: 5'- GCATGTTGCTAGGCATAACTCCTCTGCGA- 3'

The reference sequence of the relevant portion of the human COL1A2 gene is given below:

1 GGCAGCAGGA GGTTTCGGCT AAGTTGGAGG TACTGGCCAC GACTGCATGC

51 CCGCGCCCGC CAGGTGATAC CTCCGCCGGT GACCCAGGGG CTCTGCGACA

101 CAAGGAGTCT GCATGTCTAA GTGCTAGACA TGCTCAGCTT TGTGGATACG

151 CGGACTTTGT TGCTCCTCGC AGTCCAGTTA TGCCTAGCAA CATGCCAATC

201 TTTACAAGAG GAAACTGTAA GAAAGGGCCC AGCCGGAGAT AGAGGACCAC

The cleavage site for EcoNI is: 5' - CCTNNNNNAGG - 3'

3' - GGANNNNNTCC - 5'

The first amino acid of the COL1A2 protein is methionine, specified by the codonat nucleotides 130 - 132.

Questions

For clarity and full marks, whenever relevant:

• Use Courier New Font for sequences.
• Clearly and consistently annotate and / or colour code.(e.g. highlights / coloured letters).
• Provide a key if whenever needed.
• Provide numerical nucleotide and amino acid positions in your answers, when required.
• Use arrow symbols when needed (examples are provided below):

\f\f