Question

Questions? BIOTECHNOLOGY:

Describe and explain what restriction enzymes are?
what their important characteristic are, and how they work, and how they can be used for forensic applications( RFLP analysis)
Explain and understand gel electrophoresis, how does IT sort Dna by size?
You should understand what PCR is and the basic of how IT works and its forensic applications (STR analysis) of present with a gel fragment pattern, you should be able to interpret it.

Answer 1

Restriction Enzymes

The restriction enzyme is a protein produced by bacteria that cleaves the DNA at specific sites. This site is known as the restriction site.

The restriction enzymes protect the live bacteria from bacteriophages. They recognize and cleave at the restriction sites of the bacteriophage and destroy its DNA.

Characteristic

The restriction enzymes recognize short and specific nucleotide sequences in the DNA known as the recognition sequences When the restriction enzyme recognizes a DNA sequence, it hydrolyzes the bond between adjacent nucleotide and cuts through the DNA molecule.

The bacteria prevents its own DNA sequences from degradation by the addition of the methyl group at the adenine or cytosine bases within the recognition sequence with the help of enzyme methylases.

Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensible tools of recombinant DNA technology.

A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.

Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions are called recognition sequences and are randomly distributed throughout the DNA. Different bacterial species make restriction enzymes that recognize different nucleotide sequences.

When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH₃) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

RFLP analysis

The basic technique for the detection of RFLPs involves fragmenting a sample of DNA with the application of a restriction enzyme, which can selectively cleave a DNA molecule wherever a short, specific sequence is recognized in a process known as a restriction digest. The DNA fragments produced by the digest are then separated by length through a process known as agarose gel electrophoresis and transferred to a membrane via the Southern blot procedure. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe. A restriction fragment length polymorphism is said to occur when the length of a detected fragment varies between individuals, indicating non-identical sequence homologies. Each fragment length is considered an allele, whether it actually contains a coding region or not, and can be used in subsequent genetic analysis.

Examples

There are two common mechanisms by which the size of a particular restriction fragment can vary. In the first schematic, a small segment of the genome is being detected by a DNA probe (thicker line). In allele A, the genome is cleaved by a restriction enzyme at three nearby sites (triangles), but only the rightmost fragment will be detected by the probe. In allele a, restriction site 2 has been lost by a mutation, so the probe now detects the larger fused fragment running from sites 1 to 3. The second diagram shows how this fragment size variation would look on a Southern blot, and how each allele (two per individual) might be inherited in members of a family.

In the third schematic, the probe and restriction enzyme are chosen to detect a region of the genome that includes a variable number tandem repeat (VNTR) segment (boxes in schematic diagram). In allele c, there are five repeats in the VNTR, and the probe detects a longer fragment between the two restriction sites. In allele d, there are only two repeats in the VNTR, so the probe detects a shorter fragment between the same two restriction sites. Other genetic processes, such as insertions, deletions, translocations, and inversions, can also lead to polymorphisms. RFLP tests require much larger samples of DNA than do short tandem repeat (STR) tests.

Gel electrophoresis

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge

Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles.

Gel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via polymerase chain reaction (PCR), but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.

Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose's high gel strength allows for the handling of low percentage gels for the separation of large DNA fragments. Molecular sieving is determined by the size of pores generated by the bundles of agarose⁷ in the gel matrix. In general, the higher the concentration of agarose, the smaller the pore size. Traditional agarose gels are most effective at the separation of DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis⁶, which involves the application of alternating current from two different directions. In this way larger sized DNA fragments are separated by the speed at which they reorient themselves with the changes in current direction. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical driven chemical reaction. These thinner gels are of higher concentration, are run vertically and have better resolution. In modern DNA sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The use of capillary tubes allows for the application of high voltages, thereby enabling the separation of DNA fragments (and the determination of DNA sequence) quickly.

PCR

Polymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics. The vast majority of PCR methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit different temperature-dependent reactions – specifically, DNA melting and enzyme-driven DNA replication. PCR employs two main reagents – primers (which are short single strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region) and a DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.

Applications of the technique include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based phylogenies, or functional analysis of genes; diagnosis and monitoring of hereditary diseases; amplification of ancient DNA;^[4] analysis of genetic fingerprints for DNA profiling (for example, in forensic science and parentage testing); and detection of pathogens in nucleic acid tests for the diagnosis of  infectious diseases.

Forensic applications

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics:

• In its most discriminating form, genetic fingerprinting can uniquely discriminate any one person from the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and compared to that from suspects, or from a DNA database of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming or exonerating the people originally convicted.
• Forensic DNA typing has been an effective way of identifying or exonerating criminal suspects due to analysis of evidence discovered at a crime scene. The human genome has many repetitive regions that can be found within gene sequences or in non-coding regions of the genome. Specifically, up to 40% of human DNA is repetitive. There are two distinct categories for these repetitive, non-coding regions in the genome. The first category is called variable number tandem repeats (VNTR), which are 10–100 base pairs long and the second category is called short tandem repeats (STR) and these consist of repeated 2–10 base pair sections. PCR is used to amplify several well-known VNTRs and STRs using primers that flank each of the repetitive regions. The sizes of the fragments obtained from any individual for each of the STRs will indicate which alleles are present. By analyzing several STRs for an individual, a set of alleles for each person will be found that statistically is likely to be unique. Researchers have identified the complete sequence of the human genome. This sequence can be easily accessed through the NCBI website and is used in many real-life applications. For example, the FBI has compiled a set of DNA marker sites used for identification, and these are called the Combined DNA Index System (CODIS) DNA database. Using this database enables statistical analysis to be used to determine the probability that a DNA sample will match. PCR is a very powerful and significant analytical tool to use for forensic DNA typing because researchers only need a very small amount of the target DNA to be used for analysis. For example, a single human hair with attached hair follicle has enough DNA to conduct the analysis. Similarly, a few sperm, skin samples from under the fingernails, or a small amount of blood can provide enough DNA for conclusive analysis.
• Less discriminating forms of DNA fingerprinting can help in DNA paternity testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed (or ruled out).
• The PCR AMGX/AMGY design has been shown to not only facilitating in amplifying DNA sequences from a very minuscule amount of genome. However it can also be used for real time sex determination from forensic bone samples. This provides us with a powerful and effective way to determine the sex of not only ancient specimens but also current suspects in crimes.