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Chemical cleavage of mismatch (CCM)

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Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Anderson-Fabry disease, angiokeratoma corporis diffusum, ataxia telangiectasia, autoradiography, breast cancer, CCM, ceramide trihexosidosis, chemical mismatch cleavage, chemical reactivity, CMC, colorectal cancer, deoxyribonucliec acid, DNA, dystrophic epidermolysis bullosa, electrophoresis, Fabry disease, FAMA, Fanconi anemia, fluorescence-assisted mismatch analysis, hemophilia, heteroduplex, HOT chemical method, hybridization, hydroxylamine, hydroxylamine/osmium tetroxide chemical method, mutation screening, nucleotide, osmium tetroxide, PAGE, PCR, piperidine, polyacrylamide gel, polyacrylamide gel electrophoresis, polymerase chain reaction, retinitis pigmentosa, rhodopsin, Ruiter-Pompen-Wyers syndrome, single nucleotide polymorphisms, SNP, solid-phase technology, Sweeley-Klionsky disease, tetraethylamine chloride, TP53, tumor suppressor P53 gene.

Background
  • Genes, which are segments of deoxyribonucleic acid or DNA, are considered the building blocks of life because they provide instructions for all the cells in the body. Genes are located inside cells; they control an organism's development and functions by instructing cells to make new molecules (usually proteins). Proteins are organic (carbon-containing) compounds made of amino acids, and the sequence of the amino acids in a protein is defined by a gene. Proteins are required for the growth and maintenance of the body.
  • DNA is a long double-stranded molecule made up of large numbers of nucleotides. Nucleotides are considered 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, the basic forms of DNA. The two strands of DNA are complementary to each other. Complementary strands can form double-stranded structures by matching base pairs via hydrogen bonding (A with T and G with C). For example, the complementary strand for G-T-A-C is C-A-T-G.
  • Mutation and mismatch: A permanent variation in a DNA sequence of a gene is called a mutation. Genetic changes or mutations that occur in more than 1% of the general population are called polymorphisms. Some mutations and/or polymorphisms may influence the risk of the development of certain disorders/diseases. DNA mismatch is a type of DNA damage in which non-complementary bases are paired, e.g., A with C and so on. Mismatch may involve change/alteration in a single nucleotide base pair, which is called a point mutation. In point mutation, there may be a loss of a nucleotide base, substitution of one nucleotide base for another, or the insertion of an additional nucleotide base. The examples for base substitution include A/G, A/C, G/T, T/C, C/C, etc.
  • Chemical cleavage of mismatch: Chemical cleavage of mismatch (CCM), also known as chemical mismatch cleavage (CMC) or the HOT (hydroxylamine/osmium tetroxide) chemical method, is a fast and sensitive method for the detection of mismatch or mutations. CCM has the capacity to identify 95-100% of single-base mismatch or single nucleotide polymorphisms (SNPs). SNPs are DNA sequence variations or mutations that occur when a single nucleotide in the genome sequence is altered. For example, a SNP may change the DNA sequence 'AAGGCTAA' to 'ATGGCTAA.' Here, the adenine (A) base has been substituted with thymine (T). Since mutations may lead to the development of several diseases, CCM may be used to diagnose diseases caused by mutations.
  • CCM is the simplest chemical method for detecting mismatch in the gene sequence because the mismatched points can be acted upon by many chemicals and enzymes. In a mismatch, generally only two opposing bases in the DNA strands are involved with modification in the hydrogen bonding, thereby leading to increased chemical reactivity. The mismatch point may destabilize local regions of the DNA helix to create single strandedness, which facilitates the chemical to modify the bases and detect the mutations.
  • Process: The mismatched guanine or adenine reacts with a chemical, carbodiimide, while the cytosines and thymines in single stranded DNA are chemically modified by hydroxylamine and osmium tetroxide (chemical compounds), respectively. This key feature is utilized on heteroduplex DNA, which is formed by joining (annealing) complementary single stranded DNA from two different sources. Here, the heteroduplex is formed by combining the radiolabelled test DNA with normal DNA. The chemically modified bases (cytosine and thymine) at the point of mismatch base pairs are cleaved (cut) by a chemical substance called piperidine. This facilitates the identification of mismatch. The cleaved product is then analyzed on a polyacrylamide gel electrophoresis and visualized using autoradiography. Electrophoresis is a technique that uses electrical current to separate and analyze proteins and DNA, by electrical charge. Autoradiography is a technique where the probes, single stranded DNA sequences with complementary base pairs to the target DNA segment, are labeled (attached) with radioactive dye, which on exposure to X-rays can be visualized.
  • Advantages: The CCM analysis has the advantage of detecting 95-100% of the single-base mismatches (mutations) and can analyze large DNA fragment lengths (up to two kilobase or 2,000 base pairs). It also provides information about the exact location of the mutation site on a specific gene. The use of techniques such as polymerase chain reaction (PCR) and fluorescent detection methods have facilitated automation and helped in the rapid and accurate detection of mutations. PCR is an efficient and sensitive laboratory technique to amplify (produce multiple copies) a specific sequence of DNA into billions of its copies.

Methods
  • General: Deoxyribonucleic acid (DNA) is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides. 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. A permanent variation in a DNA sequence of a gene is called a mutation.
  • Chemical cleavage of mismatch (CCM) is a method used to detect a mutation (base mismatches/substitution/insertion). There are several steps involved in the process, which is described below in a sequential order.
  • Polymerase chain reaction (PCR): The DNA extracted from the sample of interest (e.g. blood or tissues such as muscle, skin) may be too little for further use in genetic analysis techniques such as CCM. This may be overcome by using PCR, an efficient and sensitive laboratory technique to amplify (produce multiple copies) a specific sequence of DNA into billions of its copies. The amplification process is done in the presence of sequence-specific oligonucleotide primers (single stranded) and Taq DNA polymerase in controlled conditions. Oligonucleotide primer is a sequence of nucleotides, usually of 20-50 bases that is complementary to a target DNA sequence and serves as a starting point for DNA replication. The complementary strand is a nucleic acid sequence that can form a double-stranded structure by matching base pairs (adenine (A) with thymine (T); guanine (G) with cytosine (C)). For example, the complementary strand for G-T-A-C is C-A-T-G. DNA polymerase is an enzyme that synthesizes new DNA strands using preexisting DNA strands as template, thereby assisting in DNA replication/multiplication. A radionucleotide or fluorescent marker is also incorporated (tagged or labeled) into the PCR product during the amplification process (radiolabeling of PCR). This assists in visualizing the PCR products at a later stage.
  • Heteroduplex formation: Heteroduplex is a DNA molecule; the two constitutive strands are derived from distinct sources and hence are likely to be mismatched. In this method, double-stranded DNA hybrids (heteroduplexes) are formed by joining target/test DNA with normal DNA to detect the mutation. Hybridization is the joining of the probe with the fragment, which allows the target molecule to be analyzed. The DNA probes are single stranded DNA sequences with complementary base pairs to the target DNA segment. The base pairing (A-T, G-C) is disrupted in cases of mutation, leading to a difference in the target DNA compared to the probe, resulting in the development of free bases.
  • Chemical modification and cleavage: The resulting free bases are chemically treated or modified to form a substrate (the material or substance on which an enzyme acts). This helps to cut (cleave) the DNA strand at or next to the mismatch site. The mismatch point is locally destabilized and can be easily acted upon by many chemicals and enzymes (chemically reactive), hence thus helps helping in modifying the mismatched bases. The chemicals commonly used include hydroxylamine and osmium tetroxide. However, because osmium tetroxide is toxic, chemicals such as potassium permanganate or tetraethylamine chloride may be used to overcome the drawback. The cytosines and thymines in DNA are easily chemically modified by hydroxylamine and osmium tetroxide, respectively. Once modification occurs, the DNA strand is cleaved by using a chemical piperidine to assist the next process, i.e., electrophoresis for the easy analysis of the product.
  • Polyacrylamide gel electrophoresis (PAGE): Electrophoresis is a technique that uses an electrical current to separate and analyze proteins, DNA, and RNA. Gel electrophoresis separates the molecules by molecular weight and charge. The product is analyzed on a polyacrylamide gel electrophoresis (PAGE) and probed/checked with specific complementary sequences to the target sequence, tagged with fluorescent markers, facilitating easy detection.
  • Polyacrylamide is a polymer (a large compound formed from several small molecules) of the chemical acrylamide. It is used to form a gel matrix in electrophoresis. Because of its capacity to separate molecules with high resolution, polyacrylamide is also used to separate nucleic acids that are small or those that differ in length by as little as one base pair. A base pair includes two nucleotide sequences located on opposite complementary DNA or RNA strands that are connected by hydrogen bonds.
  • Detection: Following electrophoresis, the fragments are then detected by a technique called hybridization that uses a radioactive probe with a complementary DNA sequence. Hybridization is the joining of the probe with the fragment, which allows the target molecule to be analyzed. The cleaved target sequences are labeled or tagged with a radioactive probe containing a DNA fragment with a complementary/matching sequence.. The radioactive DNA is then visualized by autoradiography, which develops film exposed to radiation (similar to an X-ray). Autoradiography is a technique where the probes are labeled (attached) with radioactive molecules (ethidium bromide), which on exposure to X-rays can be visualized. This helps in detecting the exact location of mutation/mismatch.
  • Solid-phase technology: Purification is done to isolate or separate the DNA fragment of interest and remove the excess enzymes and nucleotides, thereby helping to achieve more accurate results. The CCM method is time-consuming because it requires purification after each step. To overcome this drawback, researchers have improved the CCM method by immobilizing/loading sample DNA on solid supports made of silica. Chemicals such as potassium permanganate and hydroxylamine in TEAC (tetraethylammonium chloride) solution are used to modify the DNA bound to solid support. The modified DNA is then cleaved by piperidine and is removed from the solid support so that the mutations can be analyzed with electrophoresis. The solid-phase method is a fast, sensitive, and cost-effective method of detecting mutations because it eliminates the time-consuming DNA purification between reaction steps.

Research
  • Chemical cleavage of mismatch (CCM) is a method used for detecting mutation (base mismatches), which is a permanent variation in a DNA sequence of a gene. Several studies have been conducted to determine different diseases due to mutationwith genetic causes using CCM and also to improve the CCM method. Some of them have beenare explained below.
  • Fluorescence-assisted mismatch analysis (FAMA) method: FAMA uses a bifluorescent double-stranded DNA substrate to maximize the reliability of mutation-scanning procedures, which are based on cleavage of mismatches using chemicals. This procedure is similar to the CCM method except it uses a fluorescent-labeled probe instead of radioactive materials (ethidium bromide) to identify mutations. Thus, it prevents the disadvantages of using radioactive materials that may cause cancer over prolonged exposure. A probe is a single stranded DNA sequence with complementary base pairs to the target DNA segment. The complementary strand is a nucleic acid sequence that can form a double-stranded structure by matching base pairs (adenine (A); guanine (G); cytosine (C) and thymine (T)). For example, the complementary strand for G-T-A-C is C-A-T-G.
  • Colorectal cancer: Colon cancer is cancer of the large intestine (colon). Rectal cancer occurs on the last 8-10 inches of the colon. Colon and rectal cancers are often referred to together as colorectal cancers and are the second-leading cause of cancer-related deaths in the United States. Researchers conducted studies using FAMA and found that a mutation in the tumor-suppressor p53 gene (TP53) gene was associated with the development of colorectal cancer. FAMA, with chemical cleavage of mismatch, may help in the early diagnosis of cancer, allowing for the early initiation of treatment.
  • Breast cancer: In breast cancer cells in the breast tissue divide more rapidly than healthy cells and may spread through the breast tissue to other parts of the body (metastasize). Breast cancer is the second leading cause of cancer death in American women. In most cases, it is unclear what triggers abnormal cell growth in breast tissue, but it is estimated that between five and 10% of breast cancers are inherited. Scientists have conducted studies using CCM and found that mutations in the TP53 gene lead to the development of all types of cancer, including breast cancer.

Implications
  • Chemical cleavage of mismatch (CCM) is a method used for detecting genetic mutations. A permanent variation in a DNA sequence of a gene is called a mutation. Genetic changes or mutations that occur in more than 1% of the general population are called polymorphisms. Some mutations and polymorphisms may influence the risk of the development of certain disorders/diseases. Hence, CCM has been used for the screening of several inherited diseases. Inherited diseases indicate those diseases that are transmitted through genes and have been passed from parents to their offspring (children).
  • Hemophilia A: Hemophilia is an inherited bleeding disorder that affects the ability of blood to clot. Blood clots normally form after injury to the skin and allow the skin to heal normally. In patients with hemophilia, blood clots do not form properly, leading to bleeding that ranges from mild to severe. Hemophilia A is caused by defects in proteins called clotting factors (deficiency of factor VIII).
  • Symptoms of hemophilia usually appear during infancy or early childhood. Due to an inability to properly form clots, patients with hemophilia experience prolonged bleeding in response to injuries (such as scrapes and cuts) or nosebleeds. Patients with hemophilia may experience excessive bleeding after undergoing surgical or dental procedures and may also be more susceptible to bruising. Some patients with hemophilia have blood in their urine (hematuria) or stool (hematochezia). This is usually a result of internal bleeding from an organ, such as the kidney, bladder, intestines, or stomach. CCM has been used to check for mutations that may help to diagnose hemophilia. These tests may be used to confirm a diagnosis if there is a family history of hemophilia or if symptoms of hemophilia are present.
  • Fabry disease (Ruiter-Pompen-Wyers syndrome): Fabry disease is also known as Anderson-Fabry disease, Angiokeratoma angiokeratoma corporis diffusum, and ceramide trihexosidosis. Fabry disease is an inherited genetic disorder that causes fatty deposits in several organs of the body and is primarily found in males of all ethnic groups. Females can be carriers or have a milder form of the disease. A deficiency of the enzyme, alpha-galactosidase A, causes a ceramide trihexoside, a glycolipid (molecules made of sugars and fats), to accumulate in blood vessels and other tissues and organs, impairing their function. CCM may be used to detect mutations associated with the disease development and thereby assist in the diagnosis of the disease and the identification of individuals who are at an increased risk of developing the disease. A carrier is a person who harbors a specific infectious agent in the absence of visible clinical disease and serves as a potential source of infection.
  • Hereditary breast and ovarian cancer (BRCA1): The most significant risk factor for ovarian cancer is having an inherited mutation in one of two genes called breast cancer gene 1 (BRCA1) and breast cancer gene 2 (BRCA2). These genes were originally identified in families with multiple cases of breast cancer, but they are also responsible for about 5-10% of ovarian cancers. The identification of mutation by CCM helps in the early diagnosis and early initiation of treatment, providing better treatment outcomes.
  • Fanconi anemia: Fanconi anemia is an inherited disease that primarily affects the bone marrow, resulting in the decreased production of all types of blood cells (red and white). The lack of white blood cells predisposes the patient to infections, while the lack of red blood cells may result in bleeding and fatigue. Fanconi anemia is also associated with a broad variety of physical anomalies such as short height, abnormal bones (curved spine/scoliosis), changes in the reproductive system, and abnormal eyelids, ears, lungs, and heart. Since it is an inherited disease, the gene responsible for causing the disease can be easily recognized using CCM, which thereby helps in the early initiation of treatment.
  • Dystrophic epidermolysis bullosa: Dystrophic epidermolysis bullosa is a type of genetic disease that is characterized by the presence of fragile skin and blister formation. Blisters are skin erosions formed in response to friction, such as rubbing or scratching. Since it is an inherited disease, CCM helps in identifying the individuals at risk of developing the disease and it assists in early diagnosis.
  • Ataxia telangiectasia (A-T): Ataxia telangiectasia (A-T) is a rare, degenerative disease of the nervous system that damages the brain and causes motor skill development problems that ultimately may lead to death. The disease results from abnormalities in the ATM (ataxia telangiectasia mutated) gene. The DNA of a patient may be analyzed using CCM to determine whether or not the individual has the mutated gene. This test may be performed to confirm a diagnosis or to determine whether an individual is a carrier for the disease.
  • Retinitis pigmentosa: Retinitis pigmentosa refs to a group of hereditary eye disorders, all of which involve the retina, the light-sensitive nerve layer that lines the back of the eye. All diseases classified as retinitis pigmentosa cause progressive vision loss. Mutations in the rhodopsin gene are responsible for this disorder. Rhodopsin is a purplish-red pigment present in the retina, which helps in the perception of light by the eye. Researchers have used CCM and direct sequencing of DNA for the prenatal (before birth) detection of mutations leading to retinitis pigmentosa. Initial results have been reliable in the prenatal diagnosis of rhodopsin mutations. This early diagnosis can allow doctors to initiate treatment at an early stage in the affected child.

Limitations
  • One of the limitations of chemical cleavage of mismatch (CCM) is that there are multiple steps in the entire process, and if there is an error in just one of the steps, it may lead to inaccurate results. It also requires protective devices such as a fume hood for safe handling of the volatile reagents because inhalation of chemicals like osmium tetraoxide may lead to death.
  • The CCM method is time consuming because it requires purification after each step. Purification is done to isolate or separate the DNA fragment of interest and remove the excess enzymes and nucleotides to get accurate results. However, this has been overcome by modifications made to the CCM method.

Safety




Future research
  • Further study is required to improve the chemical cleavage of mismatch method (CCM) so that genes that are responsible for causing diseases can be identified and methods for disease prevention and control may consequently be developed. It is also important to understand the processes that lead to a build-up of faulty genes over a person's lifetime making them susceptible to various diseases and disorders. This knowledge may help in developing comprehensive personalized medicine for such conditions.

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

Bibliography
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