Restriction Enzymes

Find out more about restriction enzymes by viewing the animation and reading the article listed below.

 By restriction enzymes then amplified by polymerase chain reaction to make many to millions of copies of a single fragment.

this means in order for the new strands of DNA to be accepted by the cell it must be fixed inside a plasmid. The same restricting enzyme is used on the plasmid. This allows the DNA strand and plasmid to stick and join together. Once joined the phosphodiester bonds are formed by the DNA ligase on either edge of the strand. Transformation is the next stage as the newly formed plasmids are taken in by the host bacterial cells. The cells are now a DNA library and can be administered to the required organism.

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

A basic method in which we get specific genes integrated with another organism’s chromosome is as follows: Isolate the DNA from which selected gene is to be taken from and treat it with enzymes that will cut out that specific gene. These genes are then inserted into bacteria and grown into colonies being

Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA

Scientist have harvested many different types of restriction enzymes. Each type of restriction enzyme has a distinct shape and attaches to a specific part of DNA where it eventually makes the cut. Through the use of specific restriction enzymes, scientists can cut out the exact part of the bacteria’s DNA that they would like to use. Once they have a small snippet of DNA from a bacteria that is responsible for what they want to get, like size or taste, they have to get it into the organism. In order to accomplish this a vector is used. The vectors that are used are essentially viruses that have the ability to go into a living thing and insert their own DNA into them. There are other types of vectors besides viruses, but they are the most commonly used. Usually these kind of viruses are very harmful to their hosts, but scientists have removed the harmful part of the virus and instead of it inserted the desired DNA section that they

Restricting digestion in DNA This is a commonly used technique for molecular cloning and is also used to quickly check the identity of a plasmid. Restriction enzyme digestion uses naturally occurring enzymes that cleave the DNA. There are many different restriction enzymes allowing us to target a variety of DNA sequences. The materials needed include; DNA (the amount you cut depends on your application; diagnostic digests normally includes 500ng of DNA and molecular cloning often involves 1-3μg of DNA. The total reaction volume usually varies from 10-50μL yet depends on the application and by the volume of DNA to be cut).

Enzymes are applied to DNA to break it into smaller pieces which are called restriction endonucleases. These restriction endonucleases become

Gene splicing is a complex practice that utilizes naturally occurring substances in order produce a stable recombinant DNA molecule. Endonucleases, also known as restriction enzymes, are responsible for splitting DNA strands (Gene Splicing). These enzymes are very specific, only cutting the DNA strand when it recognizes a specific sequence, producing distinct DNA fragments. These fragments have one end with a single strand, a "sticky end", that will form hydrogen bonds with a complementary, foreign DNA fragment's "sticky end" (Griffiths). DNA ligase, another enzyme, permanently joins the original DNA fragments with these foreign DNA fragments, creating Recombinant DNA (Gene Splicing). The foreign DNA fragments must be cut by the same type of restriction enzyme in order to join with an original, complimentary DNA fragment (Griffths). This process was first used with bacteria due the presence of plasmids: circular DNA strands that are independent of the cell's chromosomal DNA (Pray). Extracted plasmids can be genetically modified into recombinant plasmids and placed into bacterial cells, which reproduce at high rates. These altered plasmids are in each bacterial clone, creating a population of cells with the desired genetic material that can be used for human application.

DNAzymes are made using a process called in vitro selection whereby a large group of random DNA strands are artificially selected for based on the ability to cleave RNA. The most studied DNAzyme is the 10-23. The name comes from the 10th round of in vitro selection and the 23rd round of cloning4. This is a 15 nucleotide sequence with proven enzymatic cleaving properties5. To target specific sites, RNA-recognizing sequences called guide strands that are 7-nucleotides long are attached to this main catalytic strand on each side. The 10-23 DNAzyme does not require cofactors, but in some cases they have been shown to improve enzymatic activity6,7.

Did you know that with the science of DNA manipulation, animal cadavers can be turned into insulin for diabetics? Back in the 80's scientists isolated the human gene for insulin and transferred it into bacteria. Now bacteria cultures are used to produce large amounts of human insulin. DNA manipulation is especially important in medicine, where it holds the hope of curing genetic diseases such as Huntington's, and even some types of cancer. There are several major types of DNA-manipulation enzymes used by living cells. The first type is DNA polymerase, which cells use to replicate their own DNA. The next type is DNA ligase, which joins 2 pieces of DNA to create a single piece. The third type is restriction enzymes, which appear to be made only by bacteria. Restriction enzymes are very important enzymes that are vital to our manipulation of DNA (Rapoza, DNA, 2).

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double