How Might A Crime Be Solved Using Restriction Enzymes?

Restriction enzymes are essential tools in forensic science, as they act like scissors and cut DNA at specific known sequences. In a crime scene, blood samples are first isolated from the blood, and DNA samples from several suspects are then removed individually. DNA fingerprinting is the most important contribution to forensic science, enabling police across the world to solve crimes. Modern-day DNA profiling relies on microsatellites or short tandem repeats (STRs), rather than minisatellites. DNA analysis can be used to solve crimes without suspects by storing DNA data in computer data banks.

An important technique used in such analyses is the “Southern blot”, developed by Edwin Southern in 1975. A sample of DNA is cut with a restriction enzyme, and the fragments are analyzed. Forensic DNA typing usually consists of comparing “evidence DNA” (DNA extracted from material left at a crime scene) with “suspect DNA” (DNA extracted). The restriction enzyme targets specific sites in the DNA that have varied positions in different individuals, leading to differing sizes of DNA fragments.

Suspects can be linked to crime scenes or one crime scene to another using DNA evidence from as little as saliva on a cigarette butt, skin cells on a steering wheel, or pet. Students learn about restriction enzymes and how they are used to cut DNA into fragments, examine a DNA sample, determine where the recognition sites are, and count the cytosine methyl groups in forensic DNA exhibits and reference samples for comparison.

In an experiment, the DNA from two suspects are each digested with two restriction enzymes in separate reactions and compared to crime scene samples after. Applying different restriction enzymes to the same DNA sample will generate different sets of DNA fragments, effectively, different RFLP patterns. Restriction enzymes and DNA ligase are often used to insert genes and other pieces of DNA into plasmids during DNA cloning. They offer unparalleled opportunities for diagnosing DNA sequence content and are used in fields as disparate as criminal forensics and basic research.

How do researchers use restriction enzymes?

Scientists use restriction enzymes to cut DNA molecules at specific locations and reattach different sequences to each other using an enzyme called DNA ligase, creating new, recombined DNA sequences. In the early 1950s, researchers Salvador Luria and Joe Bertani found that some bacteria were more resistant to viral infections than others, such as bacteriophages. These bacteriophage-resistant bacteria resisted the hijacking of their cell machinery by bacteriophages, leading to the discovery of restriction endonucleases (restriction enzymes).

The discovery of restriction enzymes began with a hypothesis by Werner Arber in the 1960s. He observed a dramatic change in bacteriophage DNA after it invaded these resistant strains of bacteria. Arber hypothesized that bacterial cells might express two types of enzymes: a restriction enzyme that recognizes and cuts up the foreign bacteriophage DNA and a modification enzyme that recognizes and modifies the bacterial DNA to protect it from the DNA-degrading activity of its own restriction enzyme. This prediction was confirmed in the late 1960s by Stuart Linn and Arber when they isolated a modification enzyme called methylase and a restriction enzyme responsible for bacteriophage resistance in the bacterium Escherichiacoli.

Hamilton Smith independently verified Arber’s hypothesis and elaborated on the initial discovery by Linn and Arber. He successfully purified a restriction enzyme from another bacterium, Haemophilus influenzae (H. influenzae), and showed that this enzyme cut DNA in the center of a specific six-base-pair sequence. Nathans and Danna later used Smith’s restriction enzyme to cut the 5, 000 base-pair genome of the SV40 virus, which infects monkey and human cells, and identified eleven differently-sized pieces of DNA. Nathans’ lab later showed that when the SV40 genome was digested with different combinations of restriction enzymes, the sizes of the resulting pieces could be used to deduce a physical map of the SV40 viral genome, a groundbreaking method for inferring gene sequence information.

As a result, Arber, Smith, and Nathans were jointly awarded the Nobel Prize in Physiology or Medicine in 1978.

Why do scientists use restriction enzymes?

Restriction enzyme digestion is a widely used technique in DNA cloning experiments, as it allows researchers to manipulate, analyze, and create new combinations of DNA sequences. In the early 1950s, researchers Salvador Luria and Joe Bertani discovered that some bacteria were more resistant to viral infections than others, known as bacteriophages. These bacteriophage-resistant bacteria resisted the hijacking of their cell machinery by bacteriophages, leading to the discovery of restriction endonucleases (restriction enzymes).

The discovery of restriction enzymes began with a hypothesis by Werner Arber in the 1960s. He hypothesized that bacterial cells might express two types of enzymes: a restriction enzyme that recognizes and cuts up foreign bacteriophage DNA and a modification enzyme that recognizes and modifies the bacterial DNA to protect it from the DNA-degrading activity of its own restriction enzyme. This prediction was confirmed in the late 1960s by Stuart Linn and Arber when they isolated a modification enzyme called methylase and a restriction enzyme responsible for bacteriophage resistance in the bacterium Escherichiacoli.

Hamilton Smith in 1970 verified Arber’s hypothesis and elaborated on the initial discovery by Linn and Arber. He successfully purified a restriction enzyme from another bacterium, Haemophilus influenzae (H. influenzae), and showed that it cut DNA in the center of a specific six-base-pair sequence. Nathans and Danna later used Smith’s restriction enzyme to cut the 5, 000 base-pair genome of the SV40 virus, which infects monkey and human cells, and identified eleven differently-sized pieces of DNA. Nathans’ lab later showed that when the SV40 genome was digested with different combinations of restriction enzymes, the sizes of the resulting pieces could be used to deduce a physical map of the SV40 viral genome, a groundbreaking method for inferring gene sequence information.

Restriction enzyme digest became a powerful tool for generating physical maps of a multitude of genomes, a significant breakthrough in the early stages of genome sequencing.

What can restriction enzymes be used for?

A restriction enzyme is a protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end. The use of restriction enzymes is critical to certain laboratory methods, including recombinant DNA technology and genetic engineering.

Restriction enzyme. Restriction enzymes are incredibly cool, and there are at least three thousand of them. Each one of these enzymes cuts a specific DNA sequence and doesn’t discriminate as to where the DNA comes from — bacteria, fungi, mouse, or human, snip, snip, snip.

What is a real life example of an enzyme in action?

• Practically all of the numerous and complex biochemical reactions that take place in animals, plants, and microorganisms are regulated by enzymes, and so there are many examples. Among some of the better-known enzymes are the digestive enzymes of animals. The enzyme pepsin, for example, is a critical component of gastric juices, helping to break down food particles in the stomach. Likewise, the enzyme amylase, which is present in saliva, converts starch into sugar, helping to initiate digestion.
• In medicine, the enzyme thrombin is used to promote wound healing. Other enzymes are used to diagnose certain diseases. The enzyme lysozyme, which destroys cell walls, is used to kill bacteria.
• The enzyme catalase brings about the reaction by which hydrogen peroxide is decomposed to water and oxygen. Catalase protects cellular organelles and tissues from damage by peroxide, which is continuously produced by metabolic reactions.

How many restriction enzymes are in use today?

More than 3, 600 restriction endonucleases are known which represent over 250 different specificities. Over 3, 000 of these have been studied in detail, and more than 800 of these are available commercially.

A restriction enzyme, restriction endonuclease, REase, ENase or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i. e. each strand) of the DNA double helix.

These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase ) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

More than 3, 600 restriction endonucleases are known which represent over 250 different specificities. Over 3, 000 of these have been studied in detail, and more than 800 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital tool in molecular cloning.

What happens if you only use one restriction enzyme?

Ideally, you will find two different restriction enzymes for your subcloning. It is also possible to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned in the correct orientation.

If you cannot find enzymes that meet these criteria, do not fear. You have other options, such as:

• Adding desired restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends of your oligos. This will allow you to produce a version of your insert flanked by restriction sites compatible with the recipient plasmid’s MCS. However, you still need to avoid restriction enzymes that cut within your insert.
• Adding desired restriction sites to your recipient plasmid : You can modify the MCS of your recipient plasmid using Annealed-oligo Cloning.

How can DNA be used to solve crimes?

What is DNA Fingerprinting?. DNA fingerprinting, also known as DNA profiling or genetic fingerprinting, is a powerful forensic technique used to identify and analyse an individual’s unique genetic characteristics. The process involves extracting DNA samples from biological materials and employing various molecular biology techniques, such as polymerase chain reaction (PCR) and gel electrophoresis, to create a distinct pattern of DNA fragments. This pattern, referred to as a fingerprint, is unique to each individual, except in the case of identical twins. By comparing the DNA fingerprints of different samples, forensic scientists can determine relationships, identify individuals and establish or exclude potential matches in criminal investigations, paternity cases and other areas of genetic research. DNA fingerprinting has proven to be a revolutionary tool in the field of forensic science, offering relative accuracy and reliability when determining the identity of individuals.

The Golden State Killer. DNA fingerprinting and familial testing, once revolutionary techniques in the investigator’s toolbox, haven’t always been enough to solve crimes, especially when dealing with cold cases. With the advent of new genomic technologies and the rise of direct-to-consumer testing, law enforcement officials began to search for new ways to use DNA to track down criminals.

Perhaps the most famous example of DNA solving a long-standing cold case is that of Joseph James DeAngelo, AKA The Golden State Killer. The moniker referred to an unidentified serial killer, rapist and burglar who committed a series of crimes in California in the 1970s and 1980s. The perpetrator was responsible for at least 13 murders, 50 sexual assaults and over 100 burglaries. The case remained unsolved for decades, making it one of the most notorious unsolved crime sprees in US history.

What are the applications of enzymes in forensic science?

The formation of different forms of an enzyme that are controlled by two or more alleles and in frequency exceeding 1% are known as polymorphic enzymes. Mutations lead to polymorphisms in enzymes and in turn there are variations in stability, expression, electric charge, kinetic property, and activity of an enzyme. Different variants of enzyme are there with little difference in amino acid sequence. They are also known as allozymes. These variations can be used as genetic markers in forensic science to help identify individuals and their relationships, drug metabolism and toxicological analysis, autopsy, and serology.

CYP superfamily of enzymes. It is most investigated for polymorphism; it is one of the isoenzymes of cytochrome P450(CYP). The CYP2D6 has high degree of polymorphism having 80 alleles and more than hundred types. In a research subject were categorized into four groups according to phenotype based on drug metabolism. CYP2D6 has role in 20-25% Drug metabolism. CYP2D6 has significance in forensic toxicological analysis as there are poor metabolizers of drug, because of absence or low level of CYP2D6 activity, due to that the effect of drug is very minimal.

On the other hand, 1-2% are ultra metabolizers because of high CYP2D6 activity. This can lead to high and quick metabolism of drug sample, for example, transformation of codeine to metabolite morphine thereby causing overdose of codeine. CYP3A4 has polymorphism and it might help in determining differences in drug metabolism, effect, and toxicity in all individuals because polymorphism may decrease, increase or eliminate the enzyme effect over the drug. There is a scope for research related to CYP3A4 polymorphism. 2.

How are restriction enzymes used in forensics?

Restriction enzymes are proteins that attach to DNA and are activated by restriction sequences in the DNA. These enzymes hydrolyze and destroy the bonds between nucleotides, which are inherited. DNA fingerprinting is a method used to identify DNA samples with similar restriction sequences. DNA fragments are placed in agar gel and an electric field is applied along the gel plate. As DNA fragments travel through the gel, they have the same restriction sequences occurring at the same locations along the DNA double strand.

Restriction enzymes protect bacteria from foreign DNA by cutting DNA at specific locations called restriction sites. However, the DNA of the organism is protected against its own restriction enzymes by methylases, which add methyl groups to adenine and cytosine that are part of the recognition sequences. Restriction enzymes are categorized into Type I and III, which perform both restriction and methylation by one large enzyme, and Type II and III, which cleave DNA at random sites without a recognition sequence.

What are the real life applications of restriction enzymes?

Genetic Engineering: The most popular application of restriction endonucleases is as a tool for genetic engineering. The endonuclease activity enables manipulation of the genome as well as introduction of sequences of interest in the host organism. This results in the production of the desired gene product by the host. This concept has wide range of applications in biotechnology in the production of antibiotics, antibodies, enzymes, and several secondary metabolites.

DNA mapping: DNA mapping using restriction enzymes (also known as restriction mapping) is a method to obtain structural information of the DNA fragment. In this technique the DNA is digested with a series of restriction enzymes to produce DNA fragments of various sizes. The resultant fragments are separated by agarose gel electrophoresis and the distance between the restriction enzyme sites can be estimated. This can be used to determine the structure of an unknown DNA fragment.

Gene Sequencing: A large DNA molecule is digested using restriction enzymes and the resulting fragments are processed through DNA sequencer to obtain the nucleotide sequence.

What are the 4 types of restriction enzymes?

Types of Restriction Enzymes Based on the composition, characteristics of the cleavage site, and the cofactor requirements, the restriction endonucleases are classified into four groups, Type I, II, III, and IV.