Abstract
The study aimed at isolation of DNA from human cells. DNA is the genetic material that forms the basic units for inheritance of traits from the parents to offspring during reproduction. DNA is unique for every person. Therefore, isolation of DNA has several applications such as identification of criminals through forensic investigations. The study used the chain-termination PCR instead of the standard PCR to isolate the DNA. The study used Sanger sequencing to analyze the DNA sequence. In this case, specially modified nucleotide (dNTPs) referred to as deoxyribonucleotides (ddNTPs) were used instead of the typical dNTPs used in the standard PCR. The PCR was followed by gel electrophoresis that separated the chain-terminated oligonucleotides on the basis of their sizes. In this manner, the chain-terminated oligonucleotides were moved away to from the negatively charged terminal because they were negatively charged molecules. Consequently, the chain-terminated oligonucleotides were moved closest to the positively charged terminal through the gel. The smallest DNA molecules were moved the furthest while the largest molecules were moved the shortest distance away from the negative terminal and through the gel. The results indicated a DNA sequences in which shortest fragment terminated at the first nucleotide from the 5′ terminal while the second-shortest terminated at the 2nd nucleotide from the 5′ end etc. Therefore, it was possible that reading the gel bands was in increasing size in the 5” to 3′ sequence of the initial DNA strand.
Keywords: PCR, Gel electrophoresis, Sanger sequencing.
Introduction
DNA is hereditary material found in human cells. The cells of a particular person have similar DNA. DNA is unique for each person. However, people with close would have similarities in the DNA. Human beings inherit DNA from their parents whereby a copy of DNA is contributed by the father and the other from the mother during fertilization. DNA profiling has been a major milestone in the development of evidence used in criminal justice systems. The application of genetic technology supports criminal investigations and has also been used in the provision of reliable evidence used in criminal justice courts. There is a wide range of evidence and agreement that DNA technology is a vital factor in the criminal justice system in various parts of the world (Kruse, 2015). The DNA technologies allow collecting data applied in criminal justice investigations since DNA is considered a valuable part of the judicial process. This paper is interested in explaining the standard process of DNA isolation that is used in criminal justice. The information contained in this paper provides a basic procedure for extraction and isolation of DNA from body samples that is applicable for different situations, not only in criminal justice (Hindmarsh & Prainsack, 2010).
DNA Isolation
The term DNA isolation refers to the process of extraction of DNA from different sources. The methods used in DNA isolation are dependent on the sources of DNA, size, and age of the sample. Irrespective of the different methods used in DNA isolation, there are similarities in these methods. Generally, DNA isolation methods rely on the separation of the DNA found in the cell nucleus from the cell’s other components. DNA isolation is essential for genetic analysis. Genetic analysis has several applications, such as scientific, forensic, medical uses. DNA analysis is important in studying both animal and plant cells for the diagnosis of various diseases. In the field of medicine, DNA analysis is essential in the development of medicines as well as their applications. DNA analysis is also applied in forensic investigations where it is used to identify individuals, especially rapists, accidents, and petty thieves (Rothe & Nagy, 2016).
DNA comprises double helix strands whose stability depends on the balanced interaction between the strands such as hydrogen bonds, surrounding water molecules, and stack interactions between neighboring bases. In this case, there are slight differences in the stability of DNA duplex. For instance, the genetic mutation that occurs due to errors during DNA replication can cause mismatches that lead to relative instabilities in duplexes. The instability in DNA is utilized by applying proofreading enzymes that assist in the identification of DNA mutations and replacing it with the correct order of nucleotides.
DNA isolation used several chemicals such as SDS, proteinase K, guanidinium chloride, and EDTA. Sodium Dodecyl Sulfate (SDS) was used as a negatively charged (anionic detergent). It was used in assisting the lysing of the cell during DNA extraction. Proteinase K was used to inactivate the nucleases that could have otherwise contributed to DNA degradation during the purification process.4M Guanidinium chloride was used to dissociate DNA and the protein through the precipitation method. EDTA was applied as a chelating agent in the extraction of DNA. It was used to chelate the metal ion into the enzymes as they are the cofactors that contributed to increased rates of enzyme activities. The chelation assisted in the deactivation of the enzymes such as DNase. The study applied the silica membrane-based spin-column DNA extraction method (Heikrujam, Kishor, & Mazumder, 2020).
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is an efficient method used to selective amplification of certain parts of the DNA in vitro. PCR was performed in a thermocycler and mainly involved three stages. The first stage was denaturing the DNA template using a high temperature ranging between 92 and 95 degrees Celsius. The second step included annealing the primers at the temperature range of 50 to 70 degrees Celsius. The third step involved the extension of the DNA molecules at about 72 degrees Celsius (Simoes et al., 2020).
The study used two proteinase K enzyme for isolation of the DNA from the cells. dNTPs that consisted of four basic nucleotides were added to the PCR reaction for optimized base incorporation. Short single DNA sequences called primers were used in PCR but in pairs to enhance the hybridization of the DNA sample. Primers also assisted in the identification of the region of DNA that was amplified. The PCR procedure was carried in a buffer that offered an optimal chemical environment for DNA polymerase to work. The study used a buffer with pH ranging between 8.0 that assisted in stabilizing Tris-HCl. Taq DNA polymerase required budder solution was made up of potassium ions obtained from the KCl solution. Such a buffer solution was essential in annealing the primer.
Gel Electrophoresis
Gel electrophoresis was used to separate the DNA materials according to size. The separation of the molecules occurred when the molecules were placed into a gel containing small pores. An electric field was created across the gel. The molecules moved faster or slower, depending on their size and electric charge amount (Al-Shuhaib, 2017).
Gel electrophoresis was efficient because the negatively charged molecules moved away from the electric current’s negative terminal. Notably, the small molecules moved faster than the large ones. Therefore, the size separation of the molecules was achieved as the molecules passed through the gel. The gel is analogous to the sieve in which small particles pass through while the large particles are prevented from passing through. Gel electrophoresis enabled the movement of the particles by application of inherent charges of the molecules.
The TAE buffer was used to provide the ions used to set up the electric field during gel electrophoresis. The weight-volume concentration of agarose gel in the TAE buffer was used to prepare the solution (Miller, Roman, & Norstrom, 2016). The gel electrophoresis was carried out in the following steps. In the first step, the samples were prepared for running. In this case, DNA was isolated using polymerase chain reaction and enzymatic digestion such that a solution was formed. The basic red dye was used to visualize how the sample DNA molecules moved through the gel. Secondly, the agarose TAE gel solution was prepared. A 1% solution of agarose was formed by dissolving 1g of agarose in 100mL of TAE. The percentage of agarose solution was significant in the determination of the size of DNA expected. Thirdly, the gel was cast by pouring it on the casting tray. When the gel had cooled and solidified, the gel formed a slab where a row of the wells was located at the top. The electrophoresis chamber was set-up by placing the solid gel in the chamber containing TAE buffer. The gel was well secured, such that the chamber wells were closest to the chamber’s negative electrode. The gel was loaded by putting the DNA samples into the well. A DNA ladder was loaded to act as a reference for the DNA sizes. The negative and positive leads were connected to the chamber and power supply. The power supply set up was switched on, and the electric field was created such that the negatively charged DNA samples began to move away from the negative electrode towards the positive electrode but through the gel. When the red dye in the sample DNA had migrated through the gel sufficiently, the power supply was switched off, and the gel removed then put in a solution of ethidium bromide. The ethidium bromide solution assisted in intercalates between DNA and the UV light (visible). Some of the ethidium chloride solutions were added to the agar gel solution to enhance its visibility. The ethidium bromide-stained gel was viewed in UV light and then captured on a camera.
Sanger sequencing
Sanger sequencing, also called chain termination, is used to determine the nucleotide sequence of a DNA sample. The experiment used Sanger sequencing as an alternative to the standard PCR procedure. In the Sanger sequencing method, DNA sequence applied a template obtained from a unique PCR technique called chain-termination PCR. Chain-termination PCR functioned similarly to the standard PCR. However, the chain-termination PCR had a major difference from the standard PCR because of the addition of a specially modified nucleotide (dNTPs) referred to as deoxyribonucleotides (ddNTPs). During the extension step of the standard PCR technique, the DNA polymerase combined with the dNTPs to a growing strand of DNA through the catalytic formation of a phosphodiester bond between the free 3” -OH group of previous nucleotides and the 5’-phosphate of the subsequent nucleotide (Arastehfa et al., 2019).
During the chain-termination PCR, a low ratio of the chain-terminating ddNTPs was mixed with normal dNTPs in the PCR reaction. Since ddNTPs doesn’t have the 3′-OH group necessary for the formation of phosphodiester bond formation; a DNA polymerase incorporated the ddNTP randomly; the extension ceased. The chain-termination PCR results were a large number of oligonucleotide copies of the DNA sequence needed and was terminated at the (n) length randomly by the 5’-ddNTPs.
Secondly, the chain-terminated oligonucleotides were separated based on their size through gel electrophoresis. In the gel electrophoresis, the DNA samples were loaded at the end f the gel matrix, and the electrical current was applied. The negatively charged oligonucleotide DNA samples were moved towards the positive terminal through the gel. The largest molecules moved closest while the smallest molecules moved furthest (Ullah et al.,2019).
In the third (last step), the gel analysis and defemination of the DNA sequence were done. In this case, the gel readings were used to determine the sequence of the DNA. Since DNA polymerase was responsible for only synthesizing the 5” to 3′ direction of the initial primer’s DNA, every terminal is containing the ddNTP corresponded to a particular nucleotide in the initial sequence. The shortest fragment terminated at the first nucleotide from the 5′ terminal while the second-shortest terminated at the 2nd nucleotide from the 5′ end and so on. Therefore, reading the gel bands in increasing size the 5” to 3′ sequence of the initial DNA strand was possible (Gade et al., 2016).
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