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Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms.
Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested.
In the presence of specific DNA repair enzymes, DNA fragments will reanneal or stick themselves to other fragments with cut ends that are complimentary to their own end sequence.
In this experiment, we will use restriction enzymes to cut up DNA from a small virus called Bacteriophage ?. If you saved the 1X TBE solution from the Gel Electrophoresis with Dyes activity, reuse it for this laboratory.
Fill a second pan with water and adjust it to 37°C on a hot plate while the students complete preparation of the restriction digests. Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed. If storing overnight, place trays in a container or ziploc baggie with 0.5X TBE solution so they do not dry out. Although methylene blue dye is not as sensitive as ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment.
Using good sterile technique, aliquot samples for students, being careful to keep everything on ice until ready to be used.
Enzymes should be stored in a foam container in the freezer (non frost-free if available), along with the special buffer for each enzyme. The ? DNA used in this laboratory can exist as either a linear or circular molecule, creating some confusion when interpreting restriction digest results. Since viruses have a relatively simple genome, scientists have studied their DNA and used this information to test theories and develop concepts that apply to the genetics of living organisms. Bacteriophage ? is a virus that attacks bacterial cells and is one of the most studied viruses. This experiment uses special “restriction” enzymes that act as chemical scissors to cut ? DNA into pieces. The faster moving, purplish band is bromophenol blue dye that migrates at roughly the same rate as a 300 base pair fragment of DNA. Following staining to locate the DNA, the gel is observed and the fragments appear as a pattern of bands.
Information may be provided by your teacher that details the process of isolating and analyzing these bands to create a DNA fingerprint. Set the micropipette to 4 µl and carefully add 4 µl of 10X restriction buffer to each tube. Close the microtubes and heat in a 55°C waterbath for 10 minutes then immediately place on ice for 2 minutes.
Add 2 µl of the appropriate restriction enzyme to the reaction tubes as indicated on the grid.
Close the microtube caps and make sure that all the liquid is at the bottom of the tube by tapping the bottom of the tube gently on the desk top.
The 1.0% agarose gel will be placed into the gel box with the wells at the negative (black) end of the box.
Add approximately 150 ml of 1X TBE solution to the box so that the gel will be covered with about 2 mm of buffer. Add 4 µl of loading dye to the bottom of each of the microtubes and eject the tip into the tube. Set up the electrophoresis apparatus as described in Gel Electrophoresis of Dyes - Activity 2. Wash the work area thoroughly to be sure that no stain solution is left in contact with surfaces.
Complete the activity sheet and appropriate forensics activities from either website below.
The sites at which each of the 3 different enzymes will cut lambda DNA are shown in the maps Enzymes A, B and C below. Calculate the size the resulting fragments will be after digestion and write them on the maps.
Are there as many bands in your gel as you would expect to see based on the results of your calculations? Under ideal conditions there would be 6 fragments from Enzymes A and B, and 8 fragments from Enzyme C.
Sometimes bands that are very close together in size will not be visible separately on these gels. Enzymes and, in particular, restriction enzymes have been invaluable assistants in the laboratory for decades, so it is not surprising that they have been instrumental in driving discoveries in epigenetics-related research as well.A Enzymatic manipulation of methylated DNA has seen a tremendous amount of innovation over the last 30 years, making these DNA modifying enzymes great tools to have in your freezer for both todaya€™s experiments and tomorrowa€™s innovation. Long before bisulfite conversion, and pre-dating the advent of PCR, restriction enzymes were used to differentiate between DNA samples for their DNA methylation status.
This pair of restriction enzymes has been particularly powerful as they recognize the same DNA sequence, but exhibit different sensitivities to DNA methylation modifications. Another simplified protocol that has been successful using smaller amounts of input genomic DNA samples, from both FFPE and other clinical tissue sources, is Reduced Representation Bisulfite Sequencing, or RRBS (5). This protocol uses MspI in an upstream digestion to decipher genomewide DNA methylation studies. McrBC digestion was combined with microarray analysis back in 2005, by Martienssen and colleagues (7).
In 2009, Irizarry and colleagues (8) incorporated McrBC into a new approach called Comprehensive High-throughput Arrays for Relative Methylation (CHARM). Scientists at New England Biolabs recently identified MspJI, one of the newest DNA methylation dependent restriction enzymes for DNA methylation research studies. These enzymes have been used to drive innovation in protocols over the years, but they are great tools to consider for further developing protocols that address the future experimental challenges in DNA methylation analysis. Related PostsEnzymatic DNA Methylation Analysis Enzymatic analysis are tried and true method for cost effective, reliable characterization of DNA methylation.
Our Lacto-Report construct design included an E.chromi Biobrick, as designed by Cambridge's 2009 iGEM team.


However, after a number of attempts to assemble this part into our machine using the Standard Assembly method with no success, we decided to determine if all the correct Biobrick restriction sites were present.
In Figure 1, the plasmid sample digested with Spe1 (Fermentas) resembles the undigested control. Thus, we concluded that the Spe1 restriction site was absent in the Orange E.chromi Biobrick. Suspecting that perhaps the restriction sites were not present, we tested the plasmid samples through restriction digests. As can be noted from Figure 2, the Xba1 site is not present in the Dark Green E.chromi Biobrick part, although the Spe1 and EcoR1 sites are present.
Although time would not allow for us to further characterise these parts, it must be noted that the parts themselves still functioned, as a visible colour change was easily detected in E.coli transformed with these Biobrick parts.
This study describes a novel helicase mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of bases that they recognize.
It doesn’t matter if the fragment that matches the cut end comes from the same organism or from a different one. This virus is 48,502 base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs. The restriction digestion takes place overnight and can be kept in the freezer until the next class period when it will be be used for gel electrophoresis. This will result in a thick gel so that at least 20 µl of sample can be loaded into each well. A description of how to use a micropipet can be found in Activity 2 - Gel Electrophoresis of Dyes. Methylene blue is non-toxic but will stain clothes, hands, and equipment, so always wear gloves.
They lose activity unless kept frozen; exposure to warm temperatures for even a short time will result in loss of activity. The special buffers contain the salt and pH requirements for optimal activity of each enzyme.
By heating the sample to 60°C for 3 minutes, immediately prior to electrophoresis, the hydrogen bonds holding the ends of the linear DNA together in a circle will be broken. The information from the relatively simple virus genomes has been used to test theories and develop concepts that apply to the genetics of living organisms. Each enzyme recognizes a unique sequence of 4-6 bases along the DNA strand and cuts the strand at these sites - the first step in a process called restriction mapping. The movement of the fragments will always be towards the positive electrode because DNA is a negatively charged molecule.
The slower moving aqua band is xylene cyanol, nearly equivalent to a 9000 base pair fragment.
In this experiment, we will compare our banding pattern with a predicted result shown in figure 1. When adding the droplets of buffer to the restriction tube, touch the pipette tip to the bottom of the tube.
Fill a styrofoam cup with ice, collect your DNA digestion tubes and keep on ice until needed.
Remove the comb by pulling straight up, making sure that the buffer covers the gel so that it will fill the wells and help them to retain their shape as the comb is removed.
This will break any hydrogen bonds holding the ends of the linear DNA together in a circle.
Addition of the loading dye will also stop the restriction reaction taking place in each tube. Use the tips that were left in each tube or make sure that you use a new tip for each sample if you stored the tubes overnight. If the bands are not visible because of a high background staining, place the gel in 0.1X TBE with gentle agitation, changing the buffer every 30-60 minutes until you are satisfied with the degree of destaining. The sizes were determined by comparison to a molecular ladder which has bands of known sizes when it is separated by electrophoresis at the same time as the digested ? DNA. This enabled locus-specific discrimination between methylated and unmethylated DNA sequence, ushering in a new era of epigenetics. Both of these approaches provide broad coverage of DNA methylation marks with excellent levels of sensitivity. Using McrBC, this team removed the methylated fraction of the genome, and then co-hybridized it with an undigested sample on high-density microarrays, enabling them to identify regions of differential DNA methylation status.
This approach uses high-density arrays as well, but also uses a novel a€?smoothing algorithma€? to more accurately measure methylation from raw microarray data. MspJI, covers a family of enzymes that are related to the known Mrr restriction enzymes in certain bacteria. They were discovered in Diplococcus pneumoniae in 1975, when they were considered unique because they cleaved DNA only at a methylated DNA sequence (11).
To learn more about these enzymes, or to discuss any technical specifications, NEBa€™s technical services group is always available for support.
Zheng Y., et al (2010) A unique family of Mrr-like modification-dependent restrictionA endonucleases.
We also suspected that perhaps one of the sites had been methylated, so we transformed the initial Biobrick plasmid received from the DNA distribution into a methylase negative strain of E.coli (Sure cells). E.chromi Orange digested with various restriction enzymes to confirm the presence of the Biobrick restriction sites.
The plasmid sample double-digested with EcoR1 and Spe1 (Fermentas) is linearised by cutting with EcoR1 but there are no distinct insert and plasmid bands, which is the banding pattern we expect to see after digesting with these two enzymes. Once again, although the part was excised using EcoR1 and Spe1 (Fermentas), when we attempted to clone anything in front of this part, by digestion with EcoR1 and Xba1 (Fermentas), we could not obtain any positive clones.
E.chromi Dark Green digested with various restriction enzymes to confirm the presence of the Biobrick restriction sites. The circular helicase dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single stranded DNA binding (SSB) protein.


This ability of DNA to repair itself has been utilized by scientists to introduce foreign DNA into an organism. Since the whole sequence of ? is already known we can predict where each restriction enzyme will cut and thus the expected size of the fragments that will be produced. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. Heat in the microwave for 30 seconds at a time, shaking gently each time, until the agarose is completely melted. The DNA of Bacteriophage ? is approximately 48,514 base pairs or 48.514 kilobase pairs in length while the human genome is approximately 3 billion base pairs. These smaller, specific sections of an organism’s DNA can then be studied in detail and an outline of the whole genome can be constructed. The fragments move through the gel at a rate that is determined by their size and shape, with the smallest moving the fastest.
The faster moving band must move at least 4-7 cm from the wells to achieve the best separation of DNA for analysis. Palindromes are groups of letters that read the same in both the forward and backwards orientation.
When bands are very small (500 bp or less) they may have run off the end of the gel and therefore no longer be present.
The MspJI family, first described in 2011, cleaves methylated cytosine when it is two nucleotides away from adenine or guanine, and leaves a four-base overhang on the 5a€™ side (9). DpnI and DpnII recognize the same sequence, but different methylation patterns and can be useful in instances where researchers would like to discern two sample populations based on their DNA methylation status.
Distinct DNA methylation patterns characterize differentiatedA human embryonic stem cells and developing human fetal liver. Maybe you havena€™t kept up…Genome-wide and Gene-specific Analysis of DNA Methylation EpiGenie recently reviewed the text Epigenetics by Jorg Tost. We then prepared plasmid from one of the colonies and performed a series of digests on the plasmid. The expected pattern is clearly visible in the mCherry control which was also digested with EcoR1 and Spe1 (Fermentas). After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3 end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. If the virus DNA is exposed to the restriction enzyme for only a short time, then not every restriction site will be cut by the enzyme. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. Alternatively, the solution can be heated on a hot plate, with occasional gentle shaking, until the agarose is melted. It will initially appear as a blue band, eventually resolving into two bands of different colors. When the purple dye from the loading dye is about 1 cm from the end of the gel, the power supply should be turned off and the gel box unplugged. In the case of DNA the letters are found on both the forward and the reverse strands of the DNA.
This enzyme was recently used to sequence and map DNA methylation patterns in lung fibroblast genomic DNA, highlighting its potential in genome-wide applications (10). DpnI cleaves methylated adenine to thymine in the 5a€™ to 3a€™ direction, while DpnII cleaves the same location, but only when adenine is unmethylated.
The MspJI family of modification-dependent restrictionA endonucleases for epigenetic studies. The cHDA reaction can be carried out at one temperature (25 C) for the entire process and can achieve up to 10 000 fold amplification.
The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. This would include transferring genes that will result in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area. This will result in fragments ranging in size from the smallest possible (all sites are cut) to in-between lengths (some of the sites are cut) to the longest (no sites are cut). Only use deionized water for making the 0.1X TBE buffer to make this stain since the high chlorine levels of most tap water will damage the DNA. However, in todaya€™s modern world, therea€™s more than one way to…RRBS (Reduced representation bisulfite sequencing) Want the sensitivity of WGSBS without the cost? Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb.
A single container of methylene blue dye should be all that is needed since it may be reused several times and disposed of down the sink. The complimentary bases on the opposite strand will be CTTAAG, which is the same as reading the first strand backwards! RRBS brings down the scale and cost of WGSBS by only sequencing a reduced, representative sample of the whole genome. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification.
Many enzymes recognize these types of sequences and will attach to the DNA at this site and then cut the strand between two of the bases. First the…Stay in the know with our twice monthly news update delivered to your inbox. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.



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