B4. 1201 388 AGGAAGGTGA 1261 408 1321 428 The above shows part of a cDNA sequenc

Question

B4. 1201 388 AGGAAGGTGA 1261 408 1321 428 The above shows part of a cDNA sequence of alpha ketoacid dehydrogenase and its translation to produce the wild type protein. Please answer the following concerning this sequence. The boldface TAC codon is the one that is mutated in the Amish population to AAC. The mutation is located in exon 9 of the mature mRNA. The result of the mutation is a change from a tyrosine (Y) to asparagine (N). How would you use the sequence above to carry out a dot blot or reverse dot blot DNA test for this allele? (Either method is fine.) Please outline how this would be done and what you would see for affected individuals, heterozy gous carriers, and wild type individuals? Please be as specific as possible.

Explanation / Answer

1.Reverse Dot blot - The allele specific oligonucloetide probes were immobilised onto the membrane in reverese dot blot.

Extraction of genomic DNA from the Amish Population tissue samples,

Exons to be tested( in this case exon 9) is amplified using primers specific for exon 9 and labelled (Biotinylated) by PCR.

PCR amplification and sequencing of the sequence (gene containing exon 9)

Sequencing of PCR product

Designing of oligonucleotide probes. Probs needs to be designed from the wild type sequence and the mutated sequence. Probes were mobilised on the nylon membrane .

The single stranded DNA/rxonic DNA was applied onto the membrane and allowed to hybridised with the oligonucleotide probe. Washing of the membrane after hybrisidation to remove the unbound DNA.

Development of the membrane/Detection by chemiluminiscence.

The bot which can be seen as colored dots can be seen as the positive spots. The positive spots can be seen in the wild type probe only, the mutated sequence cannot react and thus they were negative.

The position of two N(aspargine) nearly at 1246 th position marks the first AAC and 1253 rd marks the second AAC .

Sequence of the oligonucleotide probe marking the two mutant sites.

Affected individuals both affected/mutated more intense signal than wild type

Wild type both normal no mutation will be treated as negative signal

Heterozygous carrier one normal, one mutated moderate signal

Normal sequence AAACCCTACCCC TAC

Mutated sequence AAACCCAACCCCAAC

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