Phosphorimaging

Related Terms

Autoradiography, biotin labeling, chemiluminescence, colorimetry, electrophoresis, ELISA-PTT, epitope tags, fluorescent detection, FluoroTagsT tRNAs, in vitro translation, mutation detection, mutation scanning, nonisotopic detection, phosphorimaging, protein truncation test, PTT, SDS-PAGE, truncation mutations, tumor suppressor genes, Western blot.

Background

Genes, which are made of deoxyribonucleic acid (DNA), are considered the building blocks of life because they provide instructions for cells in the body. They are located inside cells and they control an organism's development and function by instructing cells to make new molecules (usually proteins). Genes are passed down from parents to their offspring (children).
DNA is a long thread-like (double-helix) molecule made up of large numbers of nucleotides. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids. Ribonucleic acid (RNA) is a nucleic acid that helps in protein synthesis, which is important for the growth and maintenance of the body. RNA is formed under the direction of DNA and both help to form amino acids, which are the building blocks of protein.
A mutation is a change in the sequence of base pairs in the DNA that makes up a gene. "Base pairs" refers to a pair of nitrogen bases such as adenine-thymine and guanine-cytosine in DNA and adenine-uracil and guanine-cytosine in RNA. A nonsense mutation may result in a truncated and, in many cases, nonfunctional protein. Some mutations may influence the risk of developing certain disorders/diseases. For example, mutations on the BRCA1/BRCA2 genes are associated with the development of breast and ovarian cancers.
Protein translation is a process by which the genetic code carried by messenger RNA (mRNA) directs the production of proteins from amino acids, the building blocks of proteins. This process is also known as RNA translation. mRNA is a form of RNA that carries information from DNA in the nucleus to the ribosomes within the cytoplasm of the cell, which are the sites of protein synthesis.
The protein truncation test (PTT) is a simple, fast, and efficient technique that is accurate provided that quality control is maintained. The technique screens for biologically relevant gene mutations (mutation detection), which cause an early stop to the process of protein translation which leads to incomplete and nonfunctional proteins. Certain incomplete and nonfunctional proteins may lead to the development of a disease/disorder. For example, the duchenne muscular dystrophy (DMD) gene provides instructions for the making of (encodes) the protein dystrophin. Dystrophin is an important component of muscles and provides structural stability. Certain mutations in the DMD gene may result in incomplete or nonfunctional dystropin, thereby causing the disease DMD, a rapidly progressive degenerating disease of muscles that is eventually fatal as a result of the weakening of respiratory and heart muscles.
PTT may have a wide diagnostic application in identifying the disease-causing mutations due to premature protein translation termination. Some of the conditions include breast cancer, cancer of the large intestine and rectum, bladder cancer, polycystic kidney disease (multiple cysts in kidney leading to kidney failure), Duchenne muscular dystrophy (DMD; a rapidly progressive degenerating disease of muscles), neurofibromatosis (growth of tumors along nerves in the skin, brain, and other parts of the body and skin pigmentation), and tuberous sclerosis (non-cancerous growth of tumors in skin, brain, kidney, and other organs).

Methods

PTT was first reported in 1931 and was used chiefly in the field of clinical research and did not have wide acceptance due to several limitations in its original method. However, with recent advances and modifications, PTT has evolved considerably from its original form and is the preferred mutation scanning technique applied on a protein level.
Protein truncation test method (PTT) is based on detection and size analysis of prematurely terminated translation of protein products resulting from in vitro (a test performed in a test tube in laboratory) transcription and translation of the coding sequences. These coding sequences are derived from the amplification (increase in number) of DNA/RNA samples by polymerase chain reaction (PCR).
Transcription is a process by which messenger RNA (mRNA) is synthesized from a DNA template (pattern) resulting in the transfer of genetic information from the DNA molecule to the mRNA. This process is also called DNA transcription. This is different from translation, which is a process by which the genetic code carried by mRNA directs the production of proteins from amino acids, the building blocks of proteins.
PCR is an efficient and sensitive laboratory technique to amplify (produce multiple copies) a specific sequence of DNA into billions of its copies in the presence of sequence specific oligonucleotide primers and DNA polymerase enzyme. An oligonucleotide primer is a sequence of nucleotides, usually of 20-50 bases that is complementary to a specific DNA sequence and serves as a starting point for DNA replication/multiplication. DNA polymerase is an enzyme that synthesizes new DNA strands using preexisting DNA strands as a template, thereby assisting in DNA replication.
Steps involved in the protein truncation testinclude:
Genomic RNA or DNA is extracted from the sample of interest (blood, cell culture) and purified. Usually RNA is used as an amplification template and DNA is used in few cases (e.g., BRCA1 gene, FAP gene, etc.). RNA samples are preferred as they contain the exons. Exons are sequences of DNA in a gene that contain information (codes) for protein formation (synthesis) that are transcribed to mRNA. Introns are intervening sequences in a gene situated between exons and they do not code for protein synthesis. During the transcription process, the introns from the DNA get removed (spliced) and the mRNA has only the exons. The mRNA with only exons is then translated into proteins.
The purified genomic DNA with large exons (>2kb or kilobase) may be amplified using PCR and genomic sequences containing RNA use reverse transcriptase PCR (RT-PCR). RT-PCR is a technique that involves the synthesis of complementary DNA (cDNA) from RNA by reverse transcription (the opposite of transcription i.e., synthesis of DNA under the direction of RNA). cDNA is a single stranded DNA that is complementary to mRNA. This is followed by the amplification of cDNA by PCR.
The nucleic acids (DNA/RNA) have two ends of the strand as 5' (5 prime) end and 3' end. The 5' is called a 'leader' end and 3' is known as a 'trailer' end. Specifically designed primers are used to incorporate a T7 RNA polymerase (RNAP) promoter at the 5' (5 prime) end, as the amplified product is further utilized for in vitro transcription and translation. T7 RNAP is an enzyme that catalyzes (helps in a chemical/biological reaction) to form a RNA copy of DNA or RNA template.
The amplified PCR or RT-PCR product is used as the starting material for in vitro translation and the resulting protein is analyzed by gel electrophoresis. Electrophoresis is a technique that uses electrical current to separate and analyze proteins (also DNA and RNA) by electrical charge. Gel electrophoresis separates the molecules by molecular weight and charge. The product is probed/checked with specific complementary sequences to the target sequence and tagged with fluorescent markers, facilitating easy detection. FluoroTagsT tRNAs are fluorescently labeled and may eliminate the need for radioactivity in research and diagnostic tests.
In the conventional PTT method, the translated protein products are analyzed by SDS-PAGE followed by autoradiography or phosphorimaging. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a protein electrophoresis method that separates the proteins based on their electric charge, molecular weight and structure (complex structure of amino acids and carbon, hydrogen, nitrogen etc.). These proteins are labeled (attached) with radioactive molecules that may be visualized by autoradiography, which develops film exposed to radiation (similar to an X-ray). Phosphorimaging is a technique similar to, but much more accurate than autoradiography, and is used to visualize the molecules and fragments labeled with radioactive molecules by analyzing them electronically with the help of a computer.
The recently modified PTT uses the ELISA (enzyme linked immunosorbent assay) based non-radioactive method to detect the truncated proteins (incomplete and nonfunctional proteins) and/or Western blotting. ELISA is a sensitive method to detect an antigen or an antibody linked to an enzyme as a marker for the detection of specific proteins. An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body. The immune response is in the form of proteins that are specific to an antigen and are known as antibodies or immune bodies. Western blotting is a technique used to identify and locate proteins based on their ability to bind to specific antibodies. The ELISA and Western blotting techniques do not use radioactive molecules, which require special handling with skilled persons and suitable precautions as they may cause cancer.
A biotinylated Lysine-tRNA is used to incorporate biotin label in the protein during the in vitro translation step. tRNA (transfer RNA) carries amino acids to the ribosome (a cell organelle that carries out protein synthesis) for protein synthesis. The biotinylated Lysine-tRNA is a process where the biotin (vitamin H) gets tagged (attached) to the truncated protein during its synthesis in translation for easy detection.
The biotin-labeled translated proteins are visualized by chemiluminescence, colorimetry, or fluorescent detection methods. The chemiluminescence method uses a chemical dye that binds with the biotin and the chemical reaction emits light, which is detected by a luminescent imager. Similarly, in the colorimetric detection method, a colored staining reagent such as BM teton may be detected by colorimeter. In the fluorescent detection method, fluorescent dyes, such as HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), are detected by ultra-violet (UV) illumination.
Advantages: The advantageous features of PTT include pinpointing the site of mutation, high sensitivity and specificity, low false positive rate, and most importantly, highlighting the disease-causing mutations. Specificity refers to the distinguishing quality or accuracy of the test to detect mutations and sensitivity indicates the probability of a test to correctly identify the mutation of single base pairs in a sequence. The false positive rate is the proportion of negative instances that were erroneously reported as being positive for a mutation.
Other techniques to detect mutations that may cause premature (earlier than predicted) termination (ending) of protein translation leading to incomplete and nonfunctional proteins include single strand conformation polymorphism (SSCP) analysis, heteroduplex analysis, and denaturing gradient gel electrophoresis (DGGE). SSCP analysis involves electrophoretic separation of single-stranded nucleic acids based on the difference in a single base pair (mutation). Heteroduplex analysis involves the mixing of test DNA and wild-type DNA (normal or reference DNA), followed by heating to melt the duplex (denature), then cooled to reanneal (joined) the duplex. Any difference between two DNA sequences leads to the formation of DNA mismatch (heteroduplex DNA), which may be detected on gel electrophoresis. The DGGE analysis involves the separation of DNA fragments based on sequence differences that result in different denaturing characteristics of the DNA, when run on a gel with increasing concentrations of chemical denaturants. These techniques are time-consuming and labor-intensive in comparison to PTT and do not pinpoint the disease-causing mutations.

Research

The protein truncation test has gained widespread use not only in the clinical and diagnostic laboratories, but also in research applications such as producing intact or truncated proteins for protein-protein interactions, protein-nucleic acid interactions, biochemical analysis, epitope mapping (using epitope tags), and various others. Epitope mapping refers to the process of identification and characterization of proteins so as to find the compatible proteins and antibodies that bond with each other.
New gene mutations are being detected in inheritable conditions such as epidermolysis bullosa (a genetic disorder with fragile skin resulting in blister formation from minor mechanical friction as in rubbing of skin) and ataxia-telangiectasia (an immune disorder characterized by abnormalities of balance, enlarged blood vessels in the eyes and skin surface, and are prone to certain cancers).

Implications

The protein truncation test (PTT) has found wide applications, especially in the detection of various disease-/disorder-causing mutations, which may help with prevention or early detection of disease. PTT may enable accurate prenatal (during pregnancy) diagnosis for families who are at high risk of having further affected children (parents carry a copy of a mutated gene and there is a chance of that mutation being passed on to their children). Some of the examples are described below.
Mutations on the BRCA1/BRCA2 genes are associated with the development of breast and ovarian cancers. Detection of these mutations may help to identify the persons who may be at risk for developing these cancers, especially among cancer prone families and in certain population groups. Most of the mutations on BRCA1 and BRCA2 genes associated with breast and ovarian cancer result in shortened and nonfunctional proteins. Detection of these truncated proteins aids in the early detection of breast and ovarian cancers.
Alterations in the APC (adenomatous polyposis coli) gene are associated with familial adenomatosis polyposis (FAP), an inherited condition where the affected person is prone to develop intestinal cancer (colon and rectum). APC protein regulates cell division and prevents cellular overgrowth (tumor suppression). Most of the mutations on the APC gene result in the production of an abnormally short, nonfunctional version of the APC protein, which may lead to polyp formations that could become cancerous. Polyps are the fleshy growth on the inside wall of the intestine. Typically, the affected person may begin to develop polyps as early as in the teen years and the polyps may become cancerous by the age of 40, unless treated. Hence, the early detection in the family members of the affected person for these altered proteins by PTT, which are associated with the development of polyps, aids in timely prevention and control of FAP.
A mutation in the DMD or dystrophin gene is associated with the development of Duchenne muscular dystrophy, a rapidly progressive degenerating disease of muscles that is eventually fatal as a result of the weakening of respiratory and heart muscles.
Inheritable polycystic kidney disease is caused by mutations in the PKD1 and PKD2 genes. These genes provide instructions for making proteins, which are essential for normal development, organization, and function of the kidneys. Early detection of these mutations may help prevent and control the disease.
The neurofibromin 1 (NF1) gene provides instructions for making neurofibromin proteins, which act as tumor suppressor proteins for uncontrolled growth of nerve cells. A mutation in the NF1 gene may lead to nonfunctional and incomplete neurofibromin protein, which is associated with the development of neurofibromatosis. Neurofibromatosis is a disease condition characterized by skin pigmentation and growth of tumors along nerves in the skin, brain, and other parts of the body that may become cancerous.
Other conditions, such as tuberous sclerosis (non-cancerous growth of tumors in skin, brain, kidney, and other organs) caused by mutations on either the TSC1 and TSC2 genes and choroideremia (degenerative disease of the eye leading to vision loss which mostly affects males), which is caused by a mutation in the CHM gene on the X chromosome that codes for the Rab escort protein 1 (REP1), may be detected and diagnosed with the help of PTT.

Limitations

Limitations of the protein truncation test (PTT) include technical issues related to the use of ribonucleic acid (RNA) and stability of the transcripts derived from the two alleles present. RNA is fragile and easily damaged or degraded by ribonucleases, a RNA cutting enzyme.
Slow readout may be another limitation; it takes a relatively long time (usually 1-2 days) for the protein migration and separation because of the use of electrophoresis followed by radioactive detection, another time-consuming procedure.
The radioactive detection materials should be handled with care. Persons using any radioisotope must be well trained in their safe use and facilities should be available for the safe disposal of radioisotopic and contaminated materials. Exposure to radiation may increase the person's susceptibility to develop cancer.
These limitations on the conventional PTT method have been overcome by the use of high throughput based PTT using biotin labeled chemiluminescence, colorimetry, or fluorescent detection methods.

Future research

The application of the protein truncation test (PTT) is not restricted to the detection and diagnosis of new mutations and their related diseases, but also may contribute towards understanding how these changes cause the disease and imparting knowledge for finding potentially effective and lasting treatments (such as gene therapy) for these disease conditions. For example, mutations in the dystrophin gene are associated with the development of Duchenne muscular dystrophy (DMD), a rapidly progressive degenerating disease of muscles that is eventually fatal as a result of the weakening of respiratory and heart muscles. Progressive research to treat this condition using gene therapy is currently being done.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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