DD analysis

Related Terms

Apoptosis, automation, autoradiography, biochemical analysis, blotting technique, cancer, cDNA, DD analysis, differential display, DNA, electrophoresis, gene, gene expression, genetic analysis, glaucoma, hybridization, messenger RNA, microarrays, mutation, northern blot, nucleotides, ovarian cancer, PAGE, PCR, polyacrylamide gel, polymerase chain reaction, polymorphism, primers, proteomics, probes, restriction fragment length polymorphism, RFLP, reverse transcription, ribonucleic acid, RNA, RT-PCR.

Background

General: Genes (deoxyribonucleic acid or DNA) are considered the building blocks of life because they provide instructions for all the cells in the body. Genes, which are located inside cells (in the nucleus specifically), control an organism's development and functions by instructing cells to make new molecules, usually proteins. Proteins are organic 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, 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.
Ribonucleic acid (RNA) is a nucleic acid that helps in protein synthesis, which is important for 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.
Differential display: The differential display (DD) technique was developed 13 years ago as a systematic method for studying eukaryotic gene expression. Eukaryotic cells are complex organized structures that are contained within a membrane. Gene expression is the conversion of the information contained in a gene first into messenger RNA and then to a protein. Messenger RNA (mRNA) is a ribonucleic acid molecule that directs protein production. The detection of any variation in the gene expression helps in determining any changes in the body, which may further develop into diseases. Hence, this technique helps in the early detection of diseases.
Differential display analysis is a widely used method for identifying differentially expressed genes. Differential gene expression is defined as a gene expression that responds to signals or triggers, such as hormones, and is a means of gene regulation by controlling (activation or inhibition of) protein synthesis. The effects of certain substances, such as hormones, on protein synthesis lead to the development of differentially expressed genes or altered gene expression. The altered gene expression may in turn lead to the development of several diseases. Hormones are chemicals released into the body by a gland or tissue that have a specific effect on tissues elsewhere in the body.
Uses: Gene expression helps to regulate or control various cellular processes, such as the development of an organism, cell cycle, programmed cell death (apoptosis, a normal process), and destructive (pathological) changes in the cell leading to the development of various diseases including cancer. Hence, monitoring the patterns of gene expression may help those in the medical field understand the various cellular/biological processes. This knowledge may further assist in the diagnosis of a disease at an early stage so that earlier initiation of treatment is possible. In the analysis of gene expression, mRNA made from many different genes in various cell types is measured. Differential display analysis is being used as a diagnostic test to help identify different types of tumors (cancer) and their subtypes, to help predict which patients may respond to treatment and which patients may be at increased risk for cancer relapse (i.e., the return of signs of a disease after a certain time period of improvement).
Process: Differential display involves the use of a combination of three frequently used molecular biology techniques: reverse transcription polymerase chain reaction (RT-PCR), polyacrylamide gel electrophoresis (PAGE), and blotting techniques. Reverse transcription polymerase chain reaction (RT-PCR)-based technology is used to produce multiple copies (amplify) of mRNA from specific cells or tissues. PAGE is performed to separate the proteins based on their sizes as this helps to compare the sample proteins with those taken from another cell or tissue using blotting techniques. Reverse transcription is a process of synthesis of DNA under the direction of RNA (using RNA as a template) that is the opposite of transcription wherein RNA are synthesized under the direction of DNA. Polymerase chain reaction (PCR), an in-vitro (outside the living organism) laboratory technique, is an automated process that generates a number of copies of a specific DNA/RNA sequence (amplification) within a short time in the laboratory (around 45 minutes) under a controlled environment using specific reagents and enzymes. It is widely used to amplify minute quantities of biologic material so as to provide adequate specimens (PCR products) for laboratory study.

Methods

General: DNA (deoxyribonucleic acid) 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. The complementary strand is a nucleic acid sequence that can form a double-stranded structure by matching base pairs (adenine (A) with guanine (G); cytosine (C) with thymine (T)); for example, the complementary strand for G-T-A-C is C-A-T-G.
RNA (ribonucleic acid) is a nucleic acid that helps in protein synthesis, which is important for 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. Messenger RNA (mRNA) is a ribonucleic acid molecule that directs protein production. Proteins are fundamental components of all living cells and include many substances, such as enzymes, hormones, and antibodies, which are necessary for the proper functioning of an organism.
Differential display (DD) is a widely used method for identifying differentially expressed genes. The effects of certain substances like hormones on protein biosynthesis leads to the development of differentially expressed genes. The DD analysis utilizes a combination of three frequently used molecular biology techniques, which is described below: reverse transcription (RT) of messenger ribonucleic acid (mRNA) followed by polymerase chain reaction (PCR) of the resulting complementary deoxyribonucleic acid (cDNA); polyacrylamide gel electrophoresis (PAGE); and blotting techniques to visualize and compare gene expression patterns between two or more samples. Other techniques include microarrays and restriction fragment length polymorphism, which are also described below.
Reverse transcription-polymerase chain reaction (RT-PCR): Reverse transcription is a process of synthesis of DNA under the direction of RNA that is the opposite of transcription where RNA is synthesized under the direction of DNA. RT-PCR is a laboratory technique consisting of two steps: (1) the conversion of a target RNA molecule into DNA or complementary DNA (cDNA) with the help of an enzyme and (2) reverse transcriptase and subsequently amplifying (produce multiple copies) the cDNA using PCR. The cDNA is a single stranded DNA that has been made from mRNA by the action of reverse transcriptase. Isolating or separating cDNAs from the sample mixture allows cDNA to develop expression vectors. An expression vector is a plasmid, yeast, or virus genome used experimentally to introduce foreign genetic material into a host cell to replicate and amplify the foreign DNA sequences as a recombinant (artificially made) molecule. This procedure is useful because the proteins of interest may be produced in high quantities. Also, this step simplifies the task of protein purification, which is an important process for analyzing the structure and function of protein.
Polymerase chain reaction (PCR): PCR is an efficient and sensitive laboratory technique used to amplify (produce multiple copies of) a specific sequence of DNA into billions of its copies in the presence of sequence-specific oligonucleotide primers and DNA polymerase enzymes. 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 template, thereby assisting in DNA replication. A radionucleotide is also incorporated into the PCR product during the amplification process (radiolabeling of PCR). This assists in visualizing the PCR products at a later stage. The amplified DNA is then run on electrophoresis to separate and study the DNA sequence.
Polyacrylamide gel electrophoresis (PAGE): Electrophoresis is a technique that uses electrical current to separate and study proteins, DNA, and RNA by electrical charge. Gel electrophoresis separates the molecules by molecular weight and charge. PAGE is typically used for the separation of proteins because of its small range of separation.
The sample DNAs are put on (run on) gel electrophoresis, and eventually separate based on their size and electric charges as they migrate through the gel to form distinct bands. The separated DNA fragments are then paired with (hybridized) complementary sequences of DNA called probes. The probes have specific sequences of nucleotides, which are complementary to only a specific DNA fragment (target sequence) to which they get hybridized. These special probes are tagged (attached) with a radioactive dye, facilitating detection by autoradiography. Autoradiography is a technique where the probes are labeled (attached) with radioactive molecules, which on exposure to X-rays may be visualized. The target sequence is identified with the help of fluorescent light (the radioactive dye) that is emitted.
Polyacrylamide is a large compound that is formed from several small molecules (polymers) of the chemical acrylamide and 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.
Blotting techniques: Blotting is the transfer of separated material from the gel matrix of PAGE to a solid membrane like nitrocellulose paper or nylon. This is performed after electrophoresis to help visualize the results of the separation of molecules.
Northern blot: Northern blot is used for the detection of specific RNA fragments. The cDNA or mRNA fragments of interest are separated by gel electrophoresis and blotted (transferred) on to a plastic sheet (typically nitrocellulose or nylon). Once the sample of cDNA or mRNA goes through electrophoresis, it is labeled with a radioactive probe containing a DNA fragment with a complementary/matching sequence. The fragments are then detected by a technique called hybridization that uses a radioactive probe with a matching DNA sequence. Hybridization is the joining of the probe with the fragment via hydrogen bonding, which allows the target molecule to be studied. The radioactive DNA is then visualized by autoradiography, which develops film exposed to radiation (similar to an X-ray). This technique also helps to monitor treatment in patients because the northern blot studies the change in protein expression with drug treatment.
There are other methods to explore differential gene expression by measuring the mRNAs expression such as microarrays and restriction fragment length polymorphism (RFLP), which are described below.
cDNA microarray: Gene expression microarrays may study the expression of many genes at one time. This genetic method has been used to identify new genes associated with certain diseases, to classify cancerous tumors, and to predict patient outcomes.
DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA attached. Each short piece of DNA attached to a microarray is called a probe. Every probe is different and attaches specifically to one gene. Microarrays are often designed to contain enough probes to detect hundreds or thousands of different genes. The sample cDNA is then attached to a fluorescent dye (labeled).
The labeled target is then put onto the DNA microarray. If a microarray contains a probe for a specific gene, it may be used to check for the presence of that gene in a sample. If a probe designed to detect a specific piece of target DNA or RNA is present on the microarray, then that DNA or RNA may stick to the microarray. The more DNA or RNA present in the target sample, the more it may stick to the microarray. The process of letting the target stick to the probe is called hybridization.
Researchers may measure how much of a specific cDNA was present in the biological sample by measuring the amount of fluorescent signal produced by the target that sticks to the microarray. The fluorescent signal is determined using a special machine called a plate reader that may detect and measure the strength of fluorescent light. The technique helps to detect the target sequences that may be altered and these in turn may lead to the development of several diseases. Thus, this method diagnoses the diseases accurately and therefore facilitates in taking timely treatment decisions for disease management.
Restriction fragment length polymorphism (RFLP) based-DD analysis: RFLP is a molecular laboratory technique in which the extracted cDNA from a sample is cut (cleaved) into fragments of DNA sequences by enzymes called restriction endonucleases. These enzymes cleave the DNA sequences at specific sites (recognition restriction sites) resulting in characteristic fragments of DNA of different length and strand orientation.
The fragmented DNAs are put on (run on) gel electrophoresis, which get separated based on their size and electric charges as they migrate through the gel to form distinct bands. The separated DNA fragments are then paired with complementary (hybridized) sequences of DNA called probes. The probes have specific sequences of nucleotides, which are complementary to only a specific DNA fragment (target sequence) to which they get hybridized. These special probes are tagged (attached) with a radioactive dye, facilitating detection by autoradiography. The target sequence is identified with the help of fluorescent light (the radioactive dye) that is emitted.
The RFLP analysis technique requires large quantities of sample DNA whereas PCR may amplify minute quantities of biologic material so as to provide adequate specimens (PCR products) for further analysis. With the development of PCR and its automation, certain limitations of RFLP have been overcome. Nonetheless, the advancement in techniques such as fluorescent imaging has facilitated the RFLP to become more efficient in the detection of differential gene expression or mutations (i.e., altered gene structure which increases an individual's risk to develop diseases).

Research

General: Differential display (DD) is a widely used method for identifying differentially expressed genes. Several studies have been conducted using the DD method to explore the variation in gene expression that may lead to the development of several diseases.
Glaucoma: Glaucoma is the name given to a group of conditions caused by increased intraocular (inside the eye) pressure (IOP), resulting either from a malformation or malfunction of the eye's drainage system. If left untreated, an elevated IOP may cause irreversible damage to the optic nerve (which sends visual information from the eye to the brain) resulting in a permanent loss of vision. However, early detection and treatment may slow or even halt the progression of the disease. Hence, researchers are conducting studies using DD analysis to determine if the optic nerve head (ONH) astrocytes (glial cells that provide nutrition and protection to nerve cells) lead to the development of glaucoma. Initial results show that differences in gene expression in ONH astrocytes may lead to the development and/or progression of glaucoma.
Hepatocellular carcinoma: Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in both children and adults. It starts in the hepatocytes, the main type of liver cells. Scientists studied the expression profiles of genes in patients with HCC by cDNA microarray and have identified the genes that are responsible for the cancer metastasis, i.e., the movement or transfer of disease from one part of the body to another. This may help in predicting the outcome of the disease and assist in the development of preventive strategies or initiation of treatment to prevent metastasis. Microarray is a technique that may be used to study the expression of many genes at one time and has been used to identify new genes associated with certain diseases.
Ovarian cancer: Ovarian cancer is a disease in which normal ovarian cells begin to grow in an uncontrolled, abnormal manner and produce tumors in one or both ovaries. According to the American Cancer Society, ovarian cancer ranks fifth in total cancer deaths among women. Hence, scientists have conducted studies using DD analysis along with other gene expression analysis methods and identified 40 differently expressed genes, which may be used as biomarkers for early screening and the treatment of patients with ovarian cancer. Genetic biomarkers provide an estimate of how genetic variations, also called mutations or polymorphisms, may possibly make individuals at higher risk for effects from environmental agents. These biomarkers may help to predict disease risk, outcome, and treatment response and toxicity.

Implications

General: Differential display (DD) is a widely used method for identifying differentially expressed genes and for the discovery of new genes that are involved in important biological pathways. The DD analysis is a systematic, sensitive, reproducible, and automated method of analyzing gene expression. Gene expression is the conversion of the information encoded (contained) in a gene first into messenger RNA (mRNA) and then to a protein. mRNA is a ribonucleic acid molecule that directs protein production. The DD analysis may be a convenient approach because it may allow many samples to be studied at the same time; hence, it has been used in a wide spectrum of applications.
Disease diagnosis and drug development: Gene expression helps to regulate or control various cellular processes such as the development of an organism, cell cycle, programmed cell death (apoptosis, a normal process), and destructive (pathological) changes in the cell leading to the development of several diseases including cancer. Monitoring the patterns of gene expression helps to understand the various cellular/biological processes, which may further assist in diagnosing a disease at an early stage in addition to early initiation of treatment. DD is also used in the field of drug development, which may tailor therapy for a disease, predict outcome (the course) of a disease.
Protein biomarkers/tumor markers: Genetic biomarkers provide an estimate of how genetic variations, also called mutations or polymorphisms, make individuals at higher risk for effects from environmental agents. They may help predict disease risk, outcome, and treatment response and toxicity.
The DD analysis has been used to discover new protein biomarkers, which may help in the detection and/or diagnosis of a disease, assist in monitoring the course of a disease, or may indicate the disease stage. Some of the examples include protein biomarkers or tumor markers for cancer such as breast, intestinal, skin, etc. The tumor markers may be counted and studied from very minimal biopsy material (tissue extracts), thereby creating new possibilities for monitoring cancer treatment.
Profiling/analysis of gene expression measures messenger RNA made from many different genes in various cell types. It is being used as a diagnostic test to help identify different types of tumors (cancers) and subtypes to help predict which patients may respond to treatment and which patients may be at increased risk for cancer relapse (the return of signs of a disease after a certain time of improvement).
Genetic analysis: The DD analysis helps to determine the expression of genes or proteins by cells in various biological stages to be used in biochemical or genetic analysis. Biochemical analysis is the analysis of chemical substances in living organisms such as proteins. Genetic analysis involves the study of a sample of DNA to look for mutations (changes) that may increase the risk of disease or affect the way a person responds to treatment. Hence, genetic analysis helps to develop methods that may prevent the disease development and also assist in the early initiation of treatment in affected persons to achieve better outcomes.

Limitations

Gene expression analysis by differential display (DD) is limited by the labor-intensive visual evaluation of the electrophoretic data traces. However, the recent advancements of automation (using computers) have helped overcome this limitation to an extent. Electrophoresis is a technique that uses electrical current to separate and study proteins, DNA, and RNA by electrical charge.
In microarray analysis, some fluorescent labels used for detection may alter the probe's ability to interact with the target proteins, thereby impairing the protein interactions on the microarray, which may give incorrect results. Other problems may arise as a result of improper fixing of the capture molecules to the microarray surface for the entire procedure, leading to inaccurate results.

Future research

General: Differential display (DD) is a widely used method for identifying differentially expressed genes and discovering genes that are involved in important biological pathways that may lead to disease development. Several studies are being conducted both to improve the DD analysis and screening of diseases and to develop new disease treatment strategies.
The application of differential display analysis is not restricted to detection and diagnosis of variations in gene expression but also contributes towards understanding how these changes cause the disease. This may further help scientists find effective and lasting treatments (such as gene therapy) for these disease conditions.
Proteomics: Proteomics is the study of the creation and action of proteins in the body and cells. In proteomics, two-dimensional electrophoresis has been widely used to simplify samples in order to make protein analysis easier. This technique has limited reproducibility and sensitivity. Electrophoresis is a technique that uses electrical current to separate and study proteins, DNA, and RNA by electrical charge. Hence, researchers have conducted studies using difference gel electrophoresis to give better reproducibility and sensitivity. Difference gel electrophoresis uses multiple fluorescent dyes that are attached to specific proteins of interest. Further research is required to understand the benefits and disadvantages associated with the procedure.

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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