The protein truncation test (PTT) is a mutation detection method that specifically detects mutations, which lead to the termination of mRNA translation and subsequently protein truncation. E.g.

• NONSENSE MUTATION: Single nucleotide substitution, which produces a stop codon (TGA, TAA or TAG), and mRNA translation stops at the point of mutation.
• FRAMESHIFT MUTATION: One or more nucleotides are either inserted or deleted. If the number of bases is not divisible by 3, a change in the reading frame alters the remainder of the translation of the mRNA, and frequently a stop codon is introduced prematurely.
• SPLICE SITE MUTATION: One or more nucleotides are inserted or deleted, leading to the presence of a stop codon at a splice site

PTT is very successful for mutation detection in genes where the frequency of missense mutations is low.

METHOD:

1. PCR is carried out from DNA containing the coding sequence only i.e. without introns. This can be in the form of cDNA via RT-PCR from RNA, or large exons in Genomic DNA.
2. The Essential feature of PTT is a specifically designed tailed sense primer. This contains four different regions:
• At it’s 5’ end a T7 RNA-polymerase promoter sequence, which facilitates the in vitro production of RNA
• A 5-7 bp spacer
• A eukaryotic translation initiation sequence (Kozak sequence) which includes a ATG start codon, facilitating the initiation of protein synthesis
• The 3’ region contains a gene specific sequence designed so that the sequence amplified reads in-frame from the ATG.
3. Large deletions, duplications and splicing mutations may be detected by agarose gel electrophoresis at this stage.
4. After amplification the PCR product is added to a coupled in vitro transcription-translation system e.g. Promega TNT® quick coupled transcription/translation systems. For detection a labelled amino acid is included. The label can be either a radionucleotide such as S³⁵, which is visualised by autoradiography or biotin for detection by chemiluminescence.
5. The resultant proteins are run out on a SDS-PAGE gel for sizing against normal control products and protein markers.

ADVANTAGES:

• Detects truncating mutations i.e. disease causing and not missense mutations, which often represent non-disease related sequence variation.
• Allows the analysis of large stretches of coding sequence (up to 5 kb: 2kb:genomic DNA, 1.3-1.6kb cDNA is best). Either: large single exons (DNA template) or multiple exons (RNA template). This significantly reduces workload.
• Length of the truncated protein pinpoints the position of the mutation, thereby facilitating its confirmation by sequencing analysis
• The use of RNA as a template potentially allows detection of abnormalities of message splicing.
• SENSITIVITY: In comparison with other mutation detection techniques, the sensitivity of PTT is good; detection of mutated alleles present at 5-10%
• Identification of disease genes: At a point where only segments of the sequence of a candidate gene is known, tailed primers can already be designed to scan patient samples for truncating mutations

DISADVANTAGES:

• Not applicable to all genes: dependent on the proportion of mutations that are truncating: E.g. APC, BRCA1, BRCA2 and Dystrophin all have approximately 90-95% truncating mutations, but NF1 and RTS have only 50% and 10% truncating mutations respectively.
• Most powerful as a technique when RNA is used, however, most laboratories only have DNA stored.
• The most readily available source of RNA is blood. However expression of the target gene in this tissue may be low, requiring technically more demanding nested amplification reactions to obtain sufficient signal.
• Transcripts carrying truncating mutations can be labile. This can be overcome by using DNA-based PTT.
• Cannot detect mutations occurring outside the coding region, which affect control of expression and RNA stability.
• FALSE NEGATIVES: Failure to amplify the mutated allele or failure to detect very small deletion/insertions or missense mutations. These might be:

1. Mutations in the primer binding site
2. Very small in –frame deletions/insertions because the mobility shifts are too small to detect.

   These problems can be overcome by the use of different percentage gels and overlapping primer sets.

3. Germline/somatic mosaicism
4. Large insertions, translocations and inversions which enlarge the region under analysis beyond amplifiable length.
• FALSE POSITIVES:

  Alternate splicing gives rise naturally to different size transcripts.

  Artefacts arising during RT or PCR. These usually disappear in the background of the huge amount of correctly amplified fragments, unless the errors occur in the first 1-3 rounds of amplification. Such artefacts can be recognised if two independent RT and PCR reactions are performed

REFERENCES:

Den Dunnen J T and Van Ommen G-J B (1999) The Protein Truncation Test: A Review. Human Mutation 14:95-102

Promega Literature:Mutation Detection- The Protein Truncation Test (pdf file)

**Diagram of PTT method in :

Den Dunnen J T and Van Ommen G-J B (1999) The Protein Truncation Test: A Review. Human Mutation 14:95-102

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