DNA Fingerprinting- Principle, Methods, Applications

DNA stands for deoxyribonucleic acid, and it is a complex molecule that contains all of the information necessary to build and maintain an organism. All living things have DNA within their cells, and nearly every cell in a multicellular organism possesses the full set of DNA required for that organism. However, DNA does more than specify the structure and function of living things — it also serves as the primary unit of heredity in organisms of all types. In other words, whenever organisms reproduce, a portion of their DNA is passed along to their offspring. This transmission of all or part of an organism`s DNA helps ensure a certain level of continuity from one generation to the next, while still allowing for slight changes that contribute to the diversity of life.

But what, exactly, is DNA? What smaller elements make up this complex molecule, how are these elements arranged, and how is information extracted from them? DNA is basically a long molecule that contains coded instructions for the cells. Everything the cells do is coded somehow in DNA - which cells should grow and when, which cells should die and when, which cells should make hair and what color it should be. DNA is composed of a long chain of monomer nucleotides, which are the basic units of DNA. The nucleotides of DNA consist of a deoxyribose sugar molecule to which is attached a phosphate group and one of four nitrogenous bases: two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). These four bases are often abbreviated as A, G, C and T. The nucleotides are joined together by covalent bonds between the phosphate of one nucleotide and the sugar of the next, forming a phosphate-sugar backbone from which the nitrogenous bases protrude.

The structure of DNA is a double-helix polymer, a spiral consisting of two DNA strands wound around each other. Each strand is complementary to the other, meaning that the bases on one strand can form hydrogen bonds with the bases on the other strand according to a specific rule: A pairs with T, and G pairs with C. This base pairing allows the strands to hold together and also enables the faithful replication of DNA during cell division. The sequence of bases along a strand determines the genetic information encoded in that strand. Different segments of DNA can code for different genes, which are instructions for making proteins or regulating cellular activities. The human genome, which is the complete set of DNA in a human cell, contains about 3 billion base pairs and about 20,000 genes.

DNA is essential for life as we know it. It stores, transmits and expresses the genetic information that guides the development and functioning of all living organisms. It also allows for variation and evolution through mutations and recombination. Understanding how DNA works can help us solve many problems in medicine, biotechnology, agriculture and forensics. In this article, we will explore the principle, methods and applications of DNA fingerprinting, a technique that uses DNA to identify individuals based on their unique genetic profiles.

DNA fingerprinting is based on the idea that every human being has a unique DNA sequence, except for identical twins who share the same genetic code. However, only about 0.1% of the DNA (about 3 million base pairs out of 3 billion) differs among individuals . These differences are mainly found in the non-coding regions of the DNA, which do not produce proteins but are inherited from the parents. These regions are called satellite DNA, because they can be separated from the bulk DNA by density gradient centrifugation .

Satellite DNA consists of short sequences of bases that are repeated many times in tandem (one after another). Depending on the length and number of these repeats, satellite DNA can be classified into two types: microsatellites and minisatellites . Microsatellites have 2 to 5 base pair repeats, while minisatellites have 9 to 80 base pair repeats. Both types of satellite DNA show polymorphism, which means that they vary in the number of repeats among individuals . For example, one person may have 10 repeats of a certain microsatellite, while another person may have 15 repeats of the same microsatellite. These variations are caused by mutations that occur randomly over time and are passed on to the offspring .

The principle of DNA fingerprinting is to compare the number and pattern of these repeats in different individuals and determine their genetic similarity or difference. Since each person inherits half of their satellite DNA from their father and half from their mother, they will have a unique combination of repeats that reflects their ancestry . By using specific probes that bind to certain regions of satellite DNA, it is possible to visualize these repeats on a gel or a membrane and generate a distinctive banding pattern for each individual. This pattern is called a DNA fingerprint or a DNA profile .

DNA fingerprinting can be performed by using different methods that target different types of satellite DNA. The most common methods are restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) amplification of short tandem repeats (STRs) . Both methods involve cutting the DNA with restriction enzymes, separating the fragments by electrophoresis, transferring them to a membrane, and hybridizing them with labeled probes. The main difference is that RFLP uses probes for minisatellites, while PCR uses primers for microsatellites . RFLP requires a large amount of fresh DNA sample and produces more accurate results, but it is time-consuming and costly. PCR requires a small amount of degraded DNA sample and produces faster and cheaper results, but it is less reliable and prone to contamination .

DNA fingerprinting has many applications in various fields, such as forensic science, paternity and maternity testing, personal identification, diagnosis of inherited disorders, development of cures for inherited disorders, detection of AIDS and breeding programs.

Short tandem repeats (STRs) and variable number of tandem repeats (VNTRs) are two types of DNA sequences that are repeated in tandem in the human genome. They are also known as microsatellites and minisatellites, respectively. They do not code for any proteins, but they are inherited from the parents and can vary in number and length among individuals. Therefore, they can be used as markers to identify and differentiate individuals based on their DNA profiles.

STRs are DNA sequences that consist of 2-5 base pairs (bp) that are repeated consecutively for a variable number of times. For example, the sequence AGAT may be repeated 10 times in one individual and 12 times in another individual at a specific locus on a chromosome. The number of repeats at a given locus is called an allele. STRs are usually found in the non-coding regions of the DNA, but some may also be located within genes or near gene regulatory regions. STRs are highly polymorphic, meaning that they have a high degree of variation among individuals. There are more than 10,000 STR loci in the human genome, and each locus may have up to 100 different alleles.

VNTRs are DNA sequences that consist of 9-80 bp that are repeated consecutively for a variable number of times. For example, the sequence CACGTG may be repeated 15 times in one individual and 20 times in another individual at a specific locus on a chromosome. VNTRs are also found in the non-coding regions of the DNA, and they are also highly polymorphic. There are about 3,000 VNTR loci in the human genome, and each locus may have up to 1,000 different alleles.

Both STRs and VNTRs are inherited in a Mendelian fashion, meaning that each individual inherits one allele from each parent at each locus. The combination of alleles at multiple loci forms a unique DNA profile or fingerprint for each individual. The probability of two unrelated individuals having the same DNA profile is extremely low, especially if many loci are analyzed. Therefore, STRs and VNTRs can be used to establish biological relationships, such as paternity or maternity, or to identify suspects or victims in forensic cases.

However, STRs and VNTRs also have some limitations and challenges. For example, some loci may have low variability or high mutation rates, which may reduce their usefulness or reliability. Some loci may also be affected by environmental factors or epigenetic modifications, which may alter their expression or detection. Moreover, some loci may be linked to each other or to certain genes or traits, which may raise ethical or legal issues regarding privacy or discrimination.

In summary, STRs and VNTRs are two types of DNA sequences that are repeated in tandem in the human genome. They can be used as markers to identify and differentiate individuals based on their DNA profiles. However, they also have some limitations and challenges that need to be considered when applying them in various fields.

There are two main methods of DNA fingerprinting that are widely used: restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) amplification of short tandem repeats (STRs). Both methods rely on the analysis of variable regions of DNA that are inherited and unique to each individual, except for identical twins. These regions are called minisatellites or VNTRs (variable number of tandem repeats) and microsatellites or STRs (short tandem repeats), depending on the size and number of the repeated units .

RFLP is the first method of DNA fingerprinting developed by Sir Alec Jeffreys in 1984. It involves the following steps :

• Collection of a sample of cells that contain DNA, such as blood, saliva, hair, or tissue.
• Extraction and purification of DNA from the sample.
• Digestion of DNA with restriction enzymes that cut at specific sequences, producing fragments of different lengths.
• Separation of DNA fragments by agarose gel electrophoresis, based on their size and charge.
• Transfer of separated DNA fragments from the gel to a nitrocellulose membrane by a technique called Southern blotting.
• Hybridization of the membrane with a radioactive probe that is complementary to a VNTR region of interest.
• Exposure of the membrane to an X-ray film, resulting in a pattern of dark bands that correspond to the fragments that have bound the probe.

The pattern of bands obtained by RFLP is unique for each individual and can be compared with other samples to determine identity or relatedness .

Short tandem repeats (STRs) are regions of DNA that contain 2-5 base pair repeats. They are highly variable among individuals and can be used as markers for DNA fingerprinting. STRs are amplified by a technique called polymerase chain reaction (PCR), which can produce thousands of copies of a specific DNA segment from a small amount of sample.

The steps involved in PCR amplification of STRs are:

• DNA extraction: The DNA is extracted from the sample material using chemical or enzymatic methods. The quality and quantity of the DNA are checked before proceeding to the next step.
• DNA amplification: The DNA is mixed with a buffer, a heat-stable DNA polymerase enzyme, nucleotides, and primers. Primers are short synthetic DNA sequences that are complementary to the flanking regions of the target STRs. The mixture is subjected to repeated cycles of heating and cooling in a thermal cycler machine. Each cycle consists of three steps: denaturation, annealing, and extension. In denaturation, the DNA is heated to separate the two strands. In annealing, the primers bind to their complementary sequences on the DNA strands. In extension, the DNA polymerase adds nucleotides to the 3` end of the primers, extending the DNA strands and copying the target STRs. After several cycles, millions of copies of the target STRs are generated.
• DNA separation: The amplified DNA is separated by gel electrophoresis, a technique that uses an electric field to move the DNA molecules through a porous gel matrix. The DNA molecules are stained with a dye and visualized under ultraviolet light. The size and number of the STRs determine the distance they migrate in the gel. Smaller STRs move faster and farther than larger ones.
• DNA analysis: The pattern of bands on the gel is compared with known standards or reference samples to determine the number of repeats in each STR locus. The combination of repeat numbers at different STR loci forms a unique profile for each individual. This profile can be used to identify or exclude individuals as sources of biological evidence in forensic cases, paternity tests, or other applications.

Both RFLP and PCR are powerful techniques for DNA fingerprinting, but they have different advantages and limitations depending on the type and quality of the DNA sample, the time and cost involved, and the accuracy and reliability of the results.

RFLP

RFLP stands for restriction fragment length polymorphism, which means that different individuals have different lengths of DNA fragments after cutting their DNA with restriction enzymes. These fragments can be separated by gel electrophoresis and detected by hybridizing with a radioactive probe that binds to a specific VNTR region. The pattern of bands on the X-ray film reflects the unique DNA profile of each individual.

Advantages of RFLP

• RFLP is considered to be more accurate and reliable than PCR, because it uses a larger and fresher DNA sample that is less prone to contamination or degradation.
• RFLP can detect more variations in the DNA sequence, because it uses longer VNTR regions that have more diversity among individuals.
• RFLP can also distinguish between maternal and paternal alleles, because it uses probes that are specific for each chromosome.

Limitations of RFLP

• RFLP requires a large amount of high-quality DNA sample, which may not be available or suitable for some cases, such as degraded or mixed samples.
• RFLP takes a longer time to complete, because it involves multiple steps of digestion, electrophoresis, blotting, and hybridization.
• RFLP is more costly, because it uses expensive reagents and equipment, such as restriction enzymes, radioactive probes, and X-ray film.

PCR

PCR stands for polymerase chain reaction, which means that a specific region of DNA is amplified by using primers that flank the target sequence and a polymerase enzyme that copies the DNA. The amplified DNA can be separated by gel electrophoresis and detected by staining with a dye that binds to the DNA. The number and size of the bands on the gel reflect the number and length of the STRs in each individual.

Advantages of PCR

• PCR requires a small amount of low-quality DNA sample, which can be obtained from various sources, such as hair, saliva, or semen.
• PCR takes a shorter time to complete, because it involves fewer steps of amplification and electrophoresis.
• PCR is less costly, because it uses cheaper reagents and equipment, such as primers, dyes, and thermocyclers.

Limitations of PCR

• PCR is less accurate and reliable than RFLP, because it uses a smaller and older DNA sample that is more prone to contamination or degradation.
• PCR can detect fewer variations in the DNA sequence, because it uses shorter STR regions that have less diversity among individuals.
• PCR cannot distinguish between maternal and paternal alleles, because it uses primers that are common for both chromosomes.

DNA fingerprinting is a technique for identifying and analyzing differences in DNA between individuals. It is based on DNA sequence variability and polymorphism. Some of its applications include:

• In forensic science, it is used to identify prospective criminal suspects, victims, or witnesses by comparing DNA samples from crime scenes with those from known individuals or databases. It can also be used to prove paternity and establish familial ties in cases of inheritance, immigration, or adoption .
• In medicine, it is used to diagnose inherited disorders in both prenatal and newborn babies by detecting mutations or variations in specific genes. These disorders may include cystic fibrosis, hemophilia, Huntington’s disease, familial Alzheimer’s, sickle cell anemia, thalassemia, and many others . It can also help find cures for these diseases by studying the DNA fingerprints of relatives who have a history of some particular disorder and identifying DNA patterns associated with the disease.
• In organ transplantation, it is used to match tissues of organ donors with those of recipients who need transplants by analyzing the compatibility of their human leukocyte antigens (HLA), which are proteins on the surface of white blood cells that play a role in immune response.
• In personal identification, it is used to verify the identity of a person using DNA samples from their skin, hair, saliva, blood, or other body fluids. It can also be used to trace ancestry and genealogy by comparing DNA fingerprints with those from different populations or regions .
• In biotechnology, it is used to identify and protect the commercial crop and livestock types by detecting genetic modifications or variations in their DNA. It can also be used to improve the quality and yield of crops and animals by selecting desirable traits through genetic engineering or breeding programs .
• In research, it is used to study the evolutionary relationship by analyzing the similarity or difference between the DNA sequences of different species or individuals. It can also be used to detect the presence or absence of specific genes or markers in a sample of DNA .

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