The genetic material that holds human life in this world is Deoxyribo-Nucleic Acid (DNA). It is present mostly inside the nucleus of each cell in the living body. Each nucleus has a pack of chromosomes which in turn contain DNA.
The DNA molecule occurs in two strands that wind around each other forming a double helical shape. Each strand holds a backbone made of alternating sugar (deoxyribose) and phosphate groups. To the alternating sugars, each of the four typical DNA bases such as adenine, guanine, cytosine, and thymine are attached.
The two strands are held together by bonds between the bases; adenine bonds with thymine, and cytosine bonds with guanine. The sequence of the bases along the backbones serves as instructions for assembling protein and RNA molecules.
It’s a polymer of four bases–A, C, T, and G–but it allows enormous complexity to be encoded by the pattern of those bases, one after another. DNA is organized structurally into chromosomes and then wound around nucleosomes as part of those chromosomes.
Functionally, it’s organized into genes, which are pieces of DNA, which lead to observable traits. And those traits come not from the DNA itself, but actually from the RNA that is made from the DNA, or most commonly of proteins that are made from the RNA which is made from the DNA.
So the central dogma, so-called molecular biology, is that genes, which are made of DNA, are made into messenger RNAs, which are then made into proteins. But for the most part, the observable traits of eye color or height or one thing or another of individuals come from individual proteins.
Sometimes, we’re learning in the last few years they come from RNAs themselves without being made into proteins–things like microRNAs. But those still are relatively the exception for accounting for traits
The invention of our genetic material helped scientists to come up with a variety of techniques that could be used to extract, detect and analyze samples of DNA in solving crimes, paternity tests, detecting the presence of any diseases, or even for cross-breeding techniques.
Few such techniques of DNA Typing include Restriction fragment length polymorphisms(RFLP), Polymerase chain reactions(PCR), sequence polymorphisms, gel electrophoresis, and DNA sequencing.
DNA Typing Techniques
1. Restriction Fragment Length Polymorphism (RFLP)
Restriction enzymes are proteins that cut the DNA at short, specific, particular sites called restriction sites. RFLPs are differences in the length of DNA strands among the individuals, cut by the enzymes. After a segment of DNA has been cut into pieces with restriction enzymes, researchers can examine the fragments using a laboratory method called gel electrophoresis, which separates DNA fragments according to their size.
If two individuals have differences in their DNA sequences at particular restriction sites, then the restriction enzymes will cut their DNA into fragments of different lengths. There may also be differences in the number of DNA fragments observed among two or more individuals.
RFLP analysis can be used as a form of genetic testing to observe whether an individual carries a mutant gene for a disease that runs in his or her family. Most RFLP markers are codominant (both alleles in a heterozygous sample will be detected) and highly locus-specific.
An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.
The total DNA sample obtained from a matrix is extracted after dilution and digestion using methylation-sensitive enzymes. The digested DNA is then size-fractionated on a preparative agarose gel medium, and fragments ranging from 500 to 2000 bp are excised, eluted, and cloned into a plasmid vector. Digests of the plasmids are screened to check for inserts.
The strands are further analyzed using PCR. Isolation of sufficient DNA for RFLP analysis is time-consuming and labor-intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis.
Therefore, more samples can be analyzed in a shorter time. An alternative name for the technique is the Cleaved Amplified Polymorphic Sequence assay. RFLPs have been very useful to use as markers for following genomic DNA, either from humans or other animals.
2. PCR Amplification
Polymerase Chain Reaction (PCR) is one of the most widely applied techniques that helps in the analysis of DNA bands to apply in various fields like forensics, plant breeding, cross animal breeds, etc. PCR has a wide field of the procedure. The requirements for PCR include;
- Thermal Cycler: It is the PCR machine inside which the reactions take place with samples. It has a thermal block with holes for loading the sample tubes containing reaction mixtures. It has the space to load three hundred plus vials usually distinctively set in three thermal blocks, along with preloaded coding features and screen to control the operation of the machine such as time set and program set.
- DNA Polymerase: Polymerases are used in stabilizing the reaction so that the DNA would be successfully made at the high temperature required for the reaction. Hence the most important feature of a DNA polymerase is that it should be heat stable. A few of them are PFU polymerase, obtained from Pyrococcus furiosus, and TTH polymerase obtained from Thermus thermophilus. However, the aptest and common polymerase used is Taq DNA polymerase. It is obtained from Thermus Aquaticus, which possesses the capability of tolerating very high temperatures and is found in hot springs across the world. It was first invented in 1969 at the hot spring of Yellowstone National Park. It is also believed to be the most ancient form of bacteria. They are capable of making new proteins such as DNA in high temperatures without getting degenerated, due to which Taq Polymerase is made from this group of bacteria for PCR.
- DNA Primers: Primers are short sequences of nucleotides that provide a starting point for DNA synthesis. They are attached at the end of DNA strands that are being multiplied which also helps in elongation. They are usually around 20 nucleotides in length. For a nucleotide to be used as a primer it should contain certain properties:
- Deoxyribonucleic Triphosphates(dNTPs): Nucleosides bonded with three phosphate groups containing deoxyribose as the backbone are dNTPs. They are organic compounds used as the building blocks of DNA. They help in enhancing the production of new DNA strands.
- Tris Buffer: Also known as Trisaminomethane buffers, they are used in TAE and TBE variations of buffers in solutions of nucleic acids. It helps in maintaining the pH of the reaction and has a melting point of 175 degrees celsius.
- Magnesium Chloride: The salt form of magnesium enhances the activity of Taq DNA polymerase and acts as a cofactor of the reaction by increasing the rate of amplification of DNA.
- Potassium Chloride: It neutralizes the charge present on the backbone of DNA and reduces repulsion between negatively charged DNA strands, i,e the primer and the template, and stabilizes the primer-template binding.
- Bovine Serum Albumin: BSA helps in yielding results from the most impure form of sample DNA. It removes the contamination and enhances production. It also helps in the stabilization of enzymes during the storage of the samples. Sometimes Gelatin is used as an alternative to BSA or along with BSA.
PCR takes place in four different stages which include:
- Initialization: It is done only in the presence of DNA polymerase that requires manual heat activation by Hot Start PCR.
- Denaturation: The target DNA, the sample to be amplified is heated at a temperature range of 94-98 degrees celsius for one minute, leading to the separation of the double-stranded DNA into two single-stranded DNA due to the breakage of hydrogen bonds between the base pairs in the given high temperature.
- Annealing: The new strands are slowly built from the existing two template strands by attaching the primers in complementary directions(3’-5’: 5’-3’) using Taq polymerase. This is done by lowering the temperature to 50-54 degree celsius.
- Elongation: The multiple strands are now forming with one old and one new strand of DNA and the cycle is repeated thirty to forty times for obtaining around billions of copies.
PCR can be amplified using detectors to analyze the graphical result obtained and provide the applications as necessary.
3. Sequence Polymorphism
Sequence Polymorphism involves one of two or more variants of a particular DNA sequence. The most common type of polymorphism involves variation at a single base pair. Polymorphisms can also be much larger and involve long stretches of DNA. A polymorphism has to occur in at least one in 100 people.
Polymorphisms could be not just single-letter changes like a C instead of T. They could also be something more elaborate, like a whole stretch of DNA, that is either present or absent. You might call that a copy number variant; those are all polymorphisms. But this is a general term to talk about diversity in genomes in a species.
A gene is said to be polymorphic if more than one allele occupies that gene’s locus within a population. A polymorphic variant of a gene can lead to the abnormal expression or the production of an abnormal form of the protein; this abnormality may cause or be associated with the disease.
Polymorphisms can be identified in the laboratory using a variety of methods. Many methods employ PCR to amplify the sequence of a gene. Once amplified, polymorphisms and mutations in the sequence can be detected by DNA sequencing either directly or after screening for variation with a method such as single-strand polymorphism analysis.
Application of DNA Typing Techniques
- DNA analysis helps a lot in applications such as parentage and kinship tests, especially in immigration cases. A direct match between evidence and suspect in criminal casework also relies heavily on DNA typing.
- Techniques such as RFLP helps in the detection of genetic diseases such as Cystic fibrosis from an individual to a whole set of familial lineage.
- It helps in confirming certain changed DNA levels which can link a suspect to the crime scene in forensic science and paternity testing.
- It is also used widely in genetic mapping to determine recombination rates that show the genetic distance between the loci and to identify a carrier of a disease-causing mutation in a family.
- DNA polymorphism serves as a genetic marker for its location in the chromosome thus they are convenient for analysis and is used in molecular genetic studies and research works that uses analysis of DNA in a wide range of sequencing.