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Genomic library preparation: Why does the restriction enzyme not cut into the gene?

Genomic library preparation: Why does the restriction enzyme not cut into the gene?



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I am currently trying to understand creating a genomic library more profoundly. In most textbooks I read (as well as wikipedia), they mentioned that the genomic library is created by isolating the DNA and fragment it with a specific restriction enzyme that cuts approximately as many times as there are genes. However, that cannot really work, can it?

Let's say E. coli has 4000 genes with a 4,600,000 bps genome. That means I must generate fragments of more than 1150 bps in theory (if each gene is the same length and no other sequences are present). That would mean I need a restriction enzyme that cuts about 4000 times creating over 1150bps fragments. So I would either use a restriction enzyme with a recognition site of 5bps (cuts every 1024bps) or with 6bps (cuts every 4096), of course just if the base-pairs are random. Now you already see, with the first restriction enzyme I will (even in theory) cut through many genes, while with the second I might get genes of the appropriate size but I will also fragment others. Furthermore the genes, especially in more complex organisms are not spaced out equally, but may be concentrated in some areas, while in others just repetitive sequences are located. So why does every textbook mention that I can create a full genomic library with one restriction enzyme? Wouldn't it make more sense to shear many copies of DNA randomly, just as it is done for shotgun sequencing, to get a higher coverage? So my question is, how is a genomic library REALLY prepared without knowing anything about the sequence? Do you just take into account that much genes will be cut in the middle and hope for the best? It seems like a very weird strategy to amplify complete genes.

Thank you! :)


A genomic library is generated for the purpose of encapsulating the full genetic component of an organism.

You do this by fragmenting the genome with restriction enzyme that cuts at its recognition sequence. These fragments are then taken and cloned into a plasmid, so that they can then be sequenced inside the plasmid using common sequences that are found on the plasmid but not (generally) in the organism itself. The sequencing would traditionally be performed by Sanger sequencing, which has a limit to how long a sequence you can do at once - really good sequencing will get you ~1000 bp, with sequence quality tailing off after about 600 bp.

The genomic library are not intended for expression - once genes are identified, they can be sub-cloned into an expression plasmid to see what they do. So for this purpose, cutting a gene into fragments isn't a problem, as you will find the rest on another plasmid and can re-construct the full-length gene by taking the two fragments and assembling them.

The reason you use the restriction enzyme is that the sequence that it cuts at is also used for inserting it into the plasmid.

So, at this point you might be asking yourself how do you match the ends of genes (or any sequence for that matter), when they are all cut by an identical sequence?

Well, the answer that is that you use multiple restriction enzymes, either by themselves or in combinations to generate a variety of fragments cut at different sites. This will mean that once you create the libraries from these different digestions, you can sequence through the different libraries and find where the fragments overlap, and then assemble all the sequences together into the original sequence.

For instance if you look at the picture below. If you imagine the the black boxes are the same gene, and the in the top one is cut with restriction enzyme A and the bottom with B, you can see that the two restriction enzymes generate overlapping fragments, so if you sequence the fragments from both restriction enzymes, you can find where the ends in A match others in A, by looking at the fragments in B.


Difference Between Genomic Library and cDNA Library

Genomic library and CDNA library are used in gene cloning to isolate different DNAs. The key difference between these two libraries is that genomic library contains DNA fragments that express the whole genome of an organism while in cDNA library, mRNA is taken from specific cells of an organism, and then cDNA is made from that mRNA in a reaction which is catalyzed by an enzyme.

Comparison Chart

Genomic LibrarycDNA Library
DefinitionA genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of similar vectors, each containing a different insert of DNA.A CDNA library is a combination of cloned CDNA fragments inserted into a collection of host cells, which together makes some portion of the transcriptome of the organism.
ExpressionEntire genomeOnly specific genes.
SizeLargerSmaller
IntronsPresentAbsent
VectorIt uses plasmids, cosmid, lambda phage, YAC and BAC for the accommodation of large fragments.It has no introns so uses plasmids, phagemids, lambda phage to accommodate small fragments.

What is Genomic Library?

In gene cloning process, the gene of interest is copied out of DNA separated from an organism. When DNA is separated from an organism, its all genes are extracted at one time. DNA of organism contains thousands of different genes. Genetic engineer finds one specific gene which encodes the specific protein of interest. There is no one technique by which a specific gene from DNA can be found so Scientists make gene libraries to catalog the organism’s DNA. Scientists, then select the desired gene from the library. Gene library is a collection of living bacteria colonies that have been transformed with different pieces of DNA from the organism that is the source of the desired gene. DNA are extracted from organism to construct a library which is an organized form of DNA. Genomic library and cDNA are two types of gene libraries. Various techniques of gene cloning have been used as gene strategies in recombinant technology. DNA fragments are separated by cutting with specific restriction enzymes from parent DNA. These fragments are ligated into vector molecules, and the collected molecules are transferred into host cells, one molecule in each cell. The genomic library consists of introns, junk DNA, and many other fragments. In this library, DNA is broken into smaller fragments within a cell. After this, all the little parts are inserted into a vector to make a library. The genomic library contains all DNA of the entire cell and genes consists all of their introns. Genomic DNA is the translation of the whole genome. It does not code the entire part on one codon because of the size of introns. There is no splicing mechanism in a genomic library which causes difficulty in the expression of genes taken from this library.

What is cDNA Library?

cDNA library is constructed by selecting one cell or tissue type. Then mRNA is isolated from that cell or tissue. A DNA copy of mRNA molecule is made using specific enzyme reverse transcriptase enzyme. So cDNA library contains that particular DNA which is present in mRNA. No introns and no DNA sequence are present in this library. In this library, all clones are full-length. Moreover, a cDNA clone is necessary to transect cells for protein production or for cell-based assays.

Genomic Library vs. cDNA Library

  • The genomic library has been composed directly of the genomic DNA.
  • cDNA Library has been formed by using mRNA as a template.
  • Genomic library expresses entire genome of the organism.
  • cDNA library represents only genes of specific conditions.
  • Two enzymes, restriction endonucleases, and ligases are significant for genomic library construction.
  • Reverse transcriptase enzyme plays a major role in cDNA library construction.
  • Genomic library expresses the DNA of both prokaryotic and eukaryotic organisms.
  • cDNA library represents the DNA of only eukaryotic organisms.
  • A genomic library is not capable of expression in the prokaryotic organism because they possess introns and the prokaryotic organism has no machinery to process introns.
  • cDNA library is capable of genome expression in bacteria which is prokaryotic because they lack introns.
Janet White

Janet White is a writer and blogger for Difference Wiki since 2015. She has a master's degree in science and medical journalism from Boston University. Apart from work, she enjoys exercising, reading, and spending time with her friends and family. Connect with her on Twitter @Janet__White


MRNA isolation:

The cDNA is only the coding sequences complementary to the mRNA transcript of our cell or gene of our interest, henceforth, we have to isolate mRNA first for the synthesis of cDNA.

The mRNA can be isolated using the ready to use mRNA isolation kit. To isolate mRNA from the rest of the RNA, oligo dT containing column is used in the isolation process.

Once the mRNA is transcribed, the poly-A tail is added during the process called post-transcriptional modifications. And this differentiates the mRNA from the rest of the single-stranded RNAs.

The poly-A tail of the mRNA remains bounded with the oligo dT containing column. After each round of washing, all the RNAs are washed off only the mRNA remains in the column.

In the final step, the mRNA is collected in another tube by using the elution buffer.

MRNA purification:

Purified mRNA must require for the next step, for that, the mRNA is purified using the purification kit and the oligo-dT complementary nucleotides are removed from the poly-A tail by heating it gently. The purified mRNA is used for the reverse transcriptase PCR.

Selection of enzyme:

A normal polymerase can’t synthesize DNA from RNA. We need another type of polymerase for that- a reverse transcriptase polymerase.

A reverse transcriptase enzyme is a special type of polymerase isolated from the retroviruses having the power to synthesize cDNA from the mRNA.

The Avian Myeloblastomis virus reverses transcriptase and Moloney Murine Leukemia Virus reverse transcriptase are two commercially available RTs used commonly in the cDNA library preparation.

Reverse transcripase PCR:

Normal PCR is used for the synthesis of DNA from DNA while the reverse transcriptase PCR is applicable for the synthesis of DNA from the RNA template using the reverse transcriptase enzyme.

DNA is synthesized back from the transcript or mRNA but the steps of the RT-PCR are almost similar to normal conventional PCR.

Construction of library:

Now we have the amplicons of a cDNA, the cDNA is now inserted into the plasmid using restriction digestion method.

The sticky ends are generated on a plasmid which binds complementary to the sticky ends of cDNA, these sticky ends are generated using the restriction digestion method.

Depending upon the size and type of cDNA, different types of plasmids are used for constructing different cDNA libraries. The plasmid with the cDNA is now inserted into the bacteria and grown using the nutrient media under aseptic conditions.

The graphical illustration of the process of cDNA synthesis and library preparation.

What we have now in our BAC (bacterial artificial chromosome) is known as a library of cDNA or cDNA library which has all the fragments of our interest. We can use it anytime. To use it in downstream applications, plasmid DNA is first isolated and processed pas per protocol.


Creating a Genomic Library

Molecular cloning may also be used to generate a genomic library. The library is a complete (or nearly complete) copy of an organism’s genome contained as recombinant DNA plasmids engineered into unique clones of bacteria. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can be used to determine the sequence of the DNA and the function of any genes present.

One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme (Figure 5). After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the original organism’s genome, that are each separate and distinct and can each be used for further study. This makes it possible for researchers to screen these different clones to discover the one containing a gene of interest from the original organism’s genome.

Figure 5. The generation of a genomic library facilitates the discovery of the genomic DNA fragment that contains a gene of interest. (credit “micrograph”: modification of work by National Institutes of Health)

To construct a genomic library using larger fragments of genomic DNA, an E. coli bacteriophage, such as lambda, can be used as a host (Figure 6). Genomic DNA can be sheared or enzymatically digested and ligated into a pre-digested bacteriophage lambda DNA vector. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E. coli host cells on a plate. During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. coli lawn, forming a plaque. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct genomic DNA fragment. Plaques can then be screened further to look for genes of interest. One advantage to producing a library using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.

Figure 6. Recombinant phage DNA molecules are made by ligating digested phage particles with fragmented genomic DNA molecules. These recombinant phage DNA molecules are packaged into phage particles and allowed to infect a bacterial lawn. Each plaque represents a unique recombinant DNA molecule that can be further screened for genes of interest.

To focus on the expressed genes in an organism or even a tissue, researchers construct libraries using the organism’s messenger RNA (mRNA) rather than its genomic DNA. Whereas all cells in a single organism will have the same genomic DNA, different tissues express different genes, producing different complements of mRNA. For example, all human cells’ genomic DNA contains the gene for insulin, but only cells in the pancreas express mRNA directing the production of insulin. Because mRNA cannot be cloned directly, in the laboratory mRNA must be used as a template by the retroviral enzyme reverse transcriptase to make complementary DNA (cDNA). A cell’s full complement of mRNA can be reverse-transcribed into cDNA molecules, which can be used as a template for DNA polymerase to make double-stranded DNA copies these fragments can subsequently be ligated into either plasmid vectors or bacteriophage to produce a cDNA library. The benefit of a cDNA library is that it contains DNA from only the expressed genes in the cell. This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library. The focus on translated sequences means that the library cannot be used to study the sequence and structure of the genome in its entirety. The construction of a cDNA genomic library is shown in Figure 7.

Figure 7. Complementary DNA (cDNA) is made from mRNA by the retroviral enzyme reverse transcriptase, converted into double-stranded copies, and inserted into either plasmid vectors or bacteriophage, producing a cDNA library. (credit “micrograph”: modification of work by National Institutes of Health)

Think about It


Bacteria invented genetic engineering — we made it controversial

In the past 50 years, three major discoveries have fueled the field of genetic engineering. Together, they ve revolutionized medicine and agriculture. Individually, they ve sparked controversy.

Genetic engineering: the first major discovery

It s the late 1960s. Somewhere between the summer of love and the last moon landing, scientists discovered something extraordinary about bacteria. To defend themselves from viruses, bacteria evolved molecular scissors called restriction enzymes.

Restriction enzymes recognize and cut specific patterns of DNA sequences. These patterns are common, but they don t show up in the bacteria s own genes. Invading DNA that makes its way into the bacteria gets cut by restriction enzymes and disarmed.

Using restriction enzymes, scientists can cut and paste together DNA from different species. For example, by cutting and pasting the gene for human insulin into bacteria, we can use the bacteria as biofactories to produce insulin for diabetic patients.

Restriction Enzymes are like molecular scissors that cut specific sequences of DNA. Scientists use restriction enzymes to cut and paste DNA together.

Genetic engineering: applications in agriculture

Flash forward to the early 1980s. Before the war on drugs or the Challenger disaster, scientists discovered something unusual about certain soil bacteria. When these Agrobacteria infect a plant, they cut and paste a small package of DNA into the plant s genome. The package tells the plant cells to divide and make food for the bacteria.

Scientists can employ Agrobacteria to deliver packages of their own. Using restriction enzymes, they replace some of the Agrobacterium s own genes with a gene for a useful trait, such as resistance to insects or pathogens. The Agrobacterium then delivers the gene into a crop plant.

Genetic engineering reinvented: the CRISPR revolution

Then, in 2011, the same year OMG and LOL were added to the dictionary, CRISPR entered the scene. The acronym is a mouthful, but what it really means is that bacteria have an immune system that can learn.

Bacteria store a library of leftover DNA from previous invaders in repeating patterns. This legacy DNA produces a molecular message called RNA, which interacts with a specialized protein. This protein, called Cas-9, cuts invading DNA sequences that match the RNA. This system, called CRISPR-Cas9, allows bacteria to remember and disarm potential threats.

Scientists can use the cut and paste mechanisms of CRISPR-Cas9 to edit genes in all kinds of organisms very precisely.

The Cas9 enzyme is like a molecular scalpel that s guided to a very specific DNA sequence by a molecular messenger called RNA. Scientists can use this CRISPR-Cas9 system to make extremely precise edits.

Genetic engineering: prospects and protests

Each of these discoveries restriction enzymes, agrobacteria and CRISPR was stumbled upon somewhat by accident. Studying how bacteria defend themselves has helped us defend ourselves from pathogens and diseases, keep food prices low and develop superior products. But before these tools could be put to work, we had to decide how to use them.

When restriction enzymes were first discovered, scientists thought carefully about the ethics of genetic engineering. In 1975, a small contingent of experts, journalists and lay people gathered at the famous Asilomar conference to discuss what to do with this technology. After four days of heated debates, the participants issued a statement recommending cautions and safeguards. This statement informed the official guidelines issued later by the National Institute of Health.

Although insulin-producing bacteria are literally genetically modified organisms, the term GMO didn t widely circulate until two decades after the Asilomar conference. GMO-mania erupted when scientists began inserting foreign genes into plants.

The vigorous resistance to engineered plants in particular is very puzzling. Few people protest using biofactories to produce medicine. Other foods, such as cheese produced via genetic engineering, have also met with little friction. Yet there are whole organizations committed to protesting GMO crops.

People disagree about whether a plant or animal engineered using CRISPR should be considered a GMO. Historically, GMO status has only applied to transgenic organisms: those containing a gene that was not inserted by traditional breeding.

CRISPR can be used to cut and paste a whole gene into a plant just like restriction enzymes and Agrobacteria. The resulting transgenic plant would certainly be considered a GMO.

On the other hand, CRISPR can be used to make changes that are so small and subtle it would be impossible to tell whether they occurred naturally. For example, if CRISPR was used to replace a single A with a G, this change would be indistinguishable from a natural A to G mutation.

Clearly, genetic engineering is a complex issue. There are many ways to do it, and the line between the different methods can be blurry. The potential applications of genetic engineering are even more diverse.

When stakeholders first gathered at Asilomar to discuss genetic engineering, their debates focused on applications and whether the resulting products could be dangerous. Most scientists agree that debates today should also focus on specific applications of genetic engineering, not the process itself.

After all, the process is not new. Bacteria have been genetically engineering themselves and their neighbors for eons. We only caught on a few decades ago.


How Does Ligation Work?

Once the source and destination DNA has been digested and cleaned it is ready to be ligated together. This is accomplished by using the enzyme T4 DNA ligase, which joins together double-stranded breaks of compatible DNA ends. The enzyme is capable of joining both blunt and sticky ends, though the efficiency of joining together blunt ends is much lower.

Using T4 ligase is easy. Simply mix the DNA, ligase, and buffer together and let incubate. It is important at this point to consider the molar ratio of all of the components, but there are lots of tools available to help like this one. Many commercial ligases allow incubation at room temperature and can be completed in less than half an hour for sticky ligations. Once ligation is complete, a small amount of the mixture can be transformed into competent cells to complete the cloning procedure.

Restriction cloning has revolutionized molecular biology since it was first introduced. The individual steps are quite simple, however putting them all together and getting them to work can be a different story. Luckily, there are plenty of resources available, including on Bitesize Bio to help you along the way. Good luck!


Use of the micropipetter

  1. Be sure that the amount set on the pipetter is correct.
  2. Place new tip firmly on micropipetter.
  3. Depress plunger to first stop, and hold this position. This step eliminates any air in tip.
  4. Dip tip into solution to be pipetted, and draw fluid into tip by releasing plunger. Always touch pipette tip to side of tube to dislodge any small amount stuck to tip. You now have a sample inside of your pipette tip.
  5. To expel sample, touch pipette tip to inside wall of tube into which you want to empty sample. This creates a capillary effect which will help draw fluid out of tip.
  6. Slowly depress plunger to first stop and then depress to second stop to blow out last bit of fluid in tip. DO NOT release plunger before removing tip from fluid in tube. Otherwise, it will suck fluid back into tip.
  7. When taking a sample, always check for air at the tip. If it is present, put the sample back and begin again.

Digest DNA with restriction endonucleases (keep all enzymes on ice)

  1. Label four 1.5ml tubes, in which you will perform restriction digestion: "P" for Pst1 enzyme, "E" for EcoRI enzyme, "H" for HindIII enzyme, and "L" for Lambda DNA uncut.
  2. Using table below, add reagents to each tube in this order: DNA, restriction buffer, water, and enzymes last (ask for them). Use a fresh pipette tip for each different reagent. The amounts are given in microliter (millionths of a liter).
  3. Pool and mix reagents by tapping the tube bottom on counter a couple of times (or use a microcentrifuge).
  4. Incubate all reaction tubes for 30 minutes at 37º C.
  5. The tubes will go into the freezer until the next period when the electrophoresis will be done.

Casting agarose gel

  1. Set gel-casting tray into the tray apparatus, screw tight, and insert well-forming comb into space marked with red line. There is a leveling bubble which can be used to level the tray (by turning knobs at bottom).
  2. Place tray FLAT where agarose can be poured and allowed to set UNDISTURBED.
  3. Carefully pour 40 ml of agarose solution (liquified in 60º C water bath) into casting tray. Use a toothpick to move any bubbles to edges (this must be done BEFORE gel hardens).
  4. Gel will solidify within 20 minute. Do NOT move tray while agarose is solidifying.
  5. Gently remove comb, pulling it straight up and taking care not to rip wells.
  6. When solidified, remove the gel tray from the gel-casting tray and place on platform of electrophoresis box, so that comb is at negative (BLACK) cathode end. The - charged DNA fragments will migrate towards the + anode end.
  7. Fill box with TBE buffer, to level that just covers entire surface of gel by about 2 mm.
  8. Make certain that sample wells left by comb are completely submerged by buffer.
  9. The gel is now ready to load with DNA.

Loading gel with DNA

  1. Remove the 4 tubes from the fridge and pulse spin the tubes in a centrifuge or tap the tubes firmly down on the table top so that all contents go down to the bottom of the tube.
  2. Add 2 µl loading dye to each reaction tube and tap contents of tube on table top.
  3. Use pipette to load contents of each reaction tube into a separate well in gel (total of 4 wells). Set your micropipetter on 12 µl (that should be the total contents in the tube).
  4. You will need to remember the order of your tubes since there is NO way that the gel can be marked. Use a clean pipette tip for each different tube.
    • Steady pipette over well using 2 hands.
    • Expel any air first from pipette tip.
    • Dip pipette tip through buffer, positioned over the well, and slowly expel the mixture (do not punch thru bottom of gel).
  5. The loading dye contains sucrose which is heavier than the DNA. It weighs the mixture down so it will sink into the bottom of the well.

Electrophoresis

  1. The electrophoresis chamber top is placed on the chamber, the electrodes connected to power supply--anode to anode (red-red) and cathode to cathode (black-black).
  2. Power supply is turned on and voltage set---120 V. The higher the voltage, the faster the electrophoresis time. In a few minutes, you should begin to notice the loading dye moving through the gel toward the + pole (anode). We will let it run for about 1 hour.
  3. The loading dye will resolve into 2 bands of color. The faster-moving, purplish band is the dye bromophenol blue, the slower-moving, aqua band is xylene cyanol . Bromophenol blue migrates through the gel at the same rate as a DNA fragment approximately 300 base pairs long. Xylene cyanol migrates at the a rate equivalent to approximately 2,000 base pairs long.
  4. After 1 hour, the bromophenol band should be nearing the end of the gel. Turn off the power supply, disconnect the leads, and remove the top of chamber.
  5. Carefully remove the casting tray and keep it horizontal until you are ready to put into the plastic container. Slide gel easily into staining tray labeled with your group name.
  6. Add enough methylene blue DNA stain to cover the gel and place cover on it. They will sit in the dye overnight.
  7. The gels will be washed a couple of times in distilled water (standing for 10-20 min), and will refrigerate until next lab period when we will look at them.
  8. The gels can then be placed on gel support film, which binds the gel and dehydrates it, if your instructor so chooses.

Scientists have identified and purified hundreds of different types of restriction enzymes. They are named after the genus and species of the organism they were isolated from and are given a number to indicate the order in which they were found. For example, EcoRI was the first restriction enzyme isolated from Escherichia coli strain RY13, whereas HindIII was the third enzyme isolated from Haemophilus influenzae strain R d.

DNA consists of two complementary strands of nucleotides that spiral around each other in a double helix . Restriction enzymes cut through both nucleotide strands, breaking the DNA into fragments, but they don’t always do this in the same way.

SmaI is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end.

Other restriction enzymes, like EcoRI, cut through the DNA strands at nucleotides that are not exactly opposite each other. This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.


Restriction Enzyme Digests

Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence. These enzymes occur naturally in bacteria as a defense against bacteriophages - viruses that infect bacteria. Bacterial restriction enzymes cut the invading bacteriophage DNA while leaving the bacterial genomic DNA unharmed due to addition of methyl groups.

This video explains the basic principles of restriction enzymes including: how restriction enzymes are named and the types of recognition sites and overhangs that exist. Also provided is a step-by-step generalized procedure for how to set up a restriction digest including the necessary components, the order in which the mixture should be assembled, and the typical incubation temperature and time. The importance of inactivating restriction enzymes to prevent star activity is mentioned. Tips for performing multiple enzymes digests and using controls in digestion reactions are also provided.

Procedure

Restriction enzymes, or restriction endonucleases, are used in a variety of different applications in molecular biology. These enzymes recognize and cleave a specific DNA sequence, called a restriction site. The video you are about to watch provides some background information on these miraculous molecules and shows how to set up a restriction enzyme digest.

Where do restriction enzymes come from anyway? These enzymes happen to be an adaptation of bacteria that act as a defense mechanism against viruses known as bacteriophages. Thanks to the addition of methyl groups to restriction enzymes sites on bacterial DNA, restriction enzymes only recognize and cut the phage DNA, thereby preventing infection.

Restriction enzymes have some pretty weird names. For instance, HindIII, NotI, EcoRI and BamHI. The first three letters of a restriction enzyme name refers to the organism from which it was isolated. For example, the restriction enzyme EcoRI was isolated from E. coli. The fourth letter, if necessary, refers to the bacterial strain from which the enzyme was isolated. The roman numeral indicates whether it is the first, second, third, enzyme isolated from that particular organism.

Restriction enzymes recognize a sequence of nucleotides, usually four to eight base pairs long, called a recognition site. At specific nucleotides within the sequence, the enzyme will break the phosphodiester bonds in the DNA backbone. The recognition sites are usually palindromic, meaning that the sequence reads the same forwards and backwards. When the palindrome is found on complementary strands of DNA molecule it is called an inverted-repeat palindrome.

Restriction enzymes can leave different types of ends once the DNA is cleaved: sticky ends and blunt ends. Sticky ends leave 3’ and 5’ overhangs while blunt ends leave no overhangs. The type of end dictates how the DNA fragment isolated by the restriction enzyme digest will be recombined with other DNA fragments in a process known as ligation.

A restriction enzyme digest should be carefully planned. A digestion reaction typically consists of the following: deionized water, the DNA that’s going to be cut, buffer specific to the enzyme you will use, and sometimes a protein called bovine serum albumin or BSA. BSA will stabilize the reaction by preventing enzyme from sticking to the side of the container that houses the digest. Each restriction enzyme can potentially have different buffer conditions, incubation temperatures, and requirements for BSA. Suppliers of restriction enzymes will have resources that one can check to obtain all of the necessary information.

To begin setting up the digest, retrieve the restriction enzyme from the freezer or fridge. Keep the restriction enzyme on ice or a thermal resistant container to make sure there is optimal activity for future reactions. To a microfuge tube reaction components should be added in the following order. First, a volume of sterile, nuclease-free water that will yield a final reaction volume of 20μL. Then 10x Restriction Enzyme Buffer, then BSA if needed, up to 1μg of DNA, and 2-10 units of enzyme. Units are defined as the amount of enzyme required to produce a complete digest of 1μg of control DNA in 60 minutes at 37°C in a 50μL reaction volume. After, mix by vortexing and then centrifuge briefly at 12,000xg in a microcentrifuge to collect the contents at the bottom of the tube. Then incubate at an optimal temperature for your restriction enzyme, usually 37°C in a heating block for 1 to 4 hours.

Once your digest has completed, it’s a good idea to incubate the reaction mixture at 65ⶬ to heat inactivate the restriction enzymes. While restriction enzymes cut site-specifically most of the time, prolonged incubation times can lead to star activity, which is cutting at sites that are similar, but distinct from their typical digestion sites.

Following inactivation, the DNA should be run on an agarose gel to ensure that the digest was successful.

Here are a couple of helpful hints for running your digests and ensuring success.

Sometimes you may find yourself in a situation where multiple enzymes need to be used to generate a specific DNA fragment. In this case you need to check that buffer conditions and incubation temperatures are compatible between the two enzymes, if so, then you can perform a double digest and have both enzymes cut in the same reaction. Sometimes, however, you’ll find incompatibility in the reaction conditions between the two enzymes, and in this case the workaround is to use the enzymes sequentially. For example, the digest can be performed with one enzyme first, and then the buffer composition, can be altered in order to be optimal for the second enzyme. Another way to overcome buffer incompatibility and perform a sequential digest is to purify the DNA following the initial digest and then perform the second digest.

Using controls are a good way to understand why a digest might go wrong. For example, a no enzyme control will allow you to check the integrity of DNA sample and determine if exonuclease activity is present. The use of control DNA with known restriction sites allows the activity of the enzyme to be tested.

Now that we have seen how digests are carried out, lets have a look at various ways restriction enzymes can be used.

Restriction enzymes can be used diagnostically, in order to identify particular samples. By loading a digest into a specialized chip and then placing that chip into a machine called a bioanalyzer. Researchers can examine DNA fragment sizes, produced by the digest, in order to determine the authenticity of fish samples. The different banding patterns of the same gene from a given species, or in this case different species, are called restriction fragment length polymorphisms.

Restriction enzymes can also be used in subcloning to isolate a fragment of DNA from one plasmid and insert into another, so the desired fragment can be replicated using bacteria.

By employing the polymerase chain reaction, or PCR to introduce restriction sites into genes at very specific locations, restriction enzymes can be used to determine the presence of single nucleotide differences in alternate forms of the same gene, or allele. These single nucleotide polymorphisms, or SNPs, are difficult to detect with PCR and gel electrophoresis alone. With the introduction of the restriction site within the SNP, a simple digest can distinguish between the alleles.

You’ve just watched JoVE’s video on restriction enzymes. You’ve learned where these enzymes come from, been taught some basics about how they work, seen how to set up a digest, and learned how restriction enzymes can be used in molecular biology. As always, thanks for watching!


The Institute for Creation Research

The March 1998 Impact article"Cloning - What is It and Where is It Taking Us?" discussed the procedure of cloning by somatic cell transfer. In that procedure, the nucleus from a cell derived from an embryo, a fetus, or tissue of an adult is inserted into an egg from which the nucleus has been removed. After the egg develops to the appropriate stage, the embryo is inserted into the uterus of a properly prepared female and allowed to develop to term. This produces an offspring essentially genetically identical to the animal that provided the nucleus that was inserted into the egg. This article will discuss the transfer of genes from one species to another in order to endow the recipient species with beneficial properties, or to enable the recipient to produce human proteins for injection into human patients who lack a vitally important protein.

Production of Therapeutic Proteins by Gene Transfer

There are many proteins essential to good health that some people cannot produce because of genetic defects. These proteins include various blood-clotting factors causing hemophilia, insulin (resulting in diabetes), growth hormone (resulting in lack of proper growth), and other proteins, the administration of which corrects pathological conditions or results in other therapeutic benefits. The early work in this field employed bacteria. Some bacteria in a bacterial culture may contain small circular DNA molecules called plasmids. These plasmids are not part of the chromosomal DNA that is possessed by all the bacteria of the culture. As these exceptional bacterial cells reproduce by binary fission or cell division, the plasmids are transmitted to the daughter cells. They can also be transmitted to other cells by conjugation. Scientists have learned how to utilize plasmids to transfer human genes to bacterial cells. If the gene inserted into the plasmid of bacteria is the human gene for insulin, for example, the bacteria into which this gene is inserted produces human insulin.

Insertion of a DNA section into a plasmid

Scientists have identified and isolated enzymes (called restriction enzymes, or restriction endonucleases) each of which cuts genes in very specific places. More than 100 of these enzymes have been isolated. After the position of the human gene that codes for the desired therapeutic protein has been located on the chromosome, the gene is cut out using the appropriate restriction enzymes and the gene is isolated. The same restriction enzymes are used to cut out a piece of the circular DNA plasmid. Thus, the two ends of the human gene will be those that will link up with the open ends of the plasmid. An enzyme called DNA ligase is used to couple each end of the gene to the open ends of the plasmid, restoring a circular DNA molecule with the human gene replacing the piece cut out of the plasmid. These plasmids, now including the human gene, are reinserted into bacteria. These bacteria are cultured, producing large quantities of identical bacteria carrying the human gene that is reproduced along with the bacterial DNA. Furthermore, these bacteria produce the human protein coded for by the spliced human gene. The protein is isolated from the bacterial culture, purified, and injected into those patients suffering from pathological conditions because their bodies cannot produce sufficient quantities of the protein.

Before genetic engineering, these proteins had to be painstakingly isolated from tissues or blood, but since they are produced in such minute quantities, the isolation of significant quantities required the processing of large quantities of material. As a result, they were very expensive. Relatively much larger quantities were obtained from genetically altered bacterial cultures, but the cost, although less, was still high. The bioreactors (that is, the machinery required to culture large quantities of the bacteria containing the human gene) are enormously expensive and must be operated by several scientists and technical assistants. Furthermore, the proper operation of the bioreactor is sensitive to small changes in temperature and composition of the culture broth.

Fortunately, an alternative method has been developed which promises to be much less expensive and much more efficient. This method utilizes an animal, such as a pig, as the bioreactor. It required years for scientists to design, develop, and build the very expensive, difficult to operate bioreactor devised by man. God had already devised a much more efficient, much cheaper bioreactor. Scientists finally realized that it would be possible by genetic manipulation to induce a female pig, cow, or other animal to produce the desired human proteins in its milk.

Genetic engineering of a milk protein

This procedure has now been successful in both pigs and cows. Among animals, the pig has a number of advantages. Its gestation period is only four months. At 12 months of age the pig is fertile and produces large litter sizes (usually 10 to 12 piglets). A lactating pig produces 300 liters (about 315 quarts) of milk in a year. The procedure is carried out as follows:

  1. The DNA fragment (gene) that codes for the needed human protein is isolated.
  2. The DNA fragment that promotes production of proteins in mammary glands is isolated and linked or combined to the human gene.
  3. Fertilized eggs from a donor pig are obtained.
  4. The human DNA is injected into an egg in the region of the male pronucleus (DNA from the sperm before it unites with DNA of the egg) using a very slim micro pipette. The human DNA is thus incorporated into the pig nuclear DNA.
  5. The egg is implanted in the uterus of another pig and develops into a newborn female pig.
  6. The desired protein is isolated from the milk of the female pig once grown.

This procedure was carried out successfully by American scientists and the results were published in 1994. 1 The human gene they used codes for Protein C that acts to control blood clotting. They obtained one gram of Protein C from each liter of milk produced by the pig. This is 200 times the concentration found in human blood plasma. Only about one third of the Protein C obtained was biologically active. The reason for this is that many modifications of a protein must be performed in a cell after the protein chain is formed. For example, sections are cut out of the protein complex sugar groups may be attached at certain places in the protein chain and cell wall anchor groups may be added. The scientists discovered that a key processing enzyme, called furin, was present in insufficient quantities, so they added to their gene complex the gene that codes for furin. This increased the yield of active Protein C. The human proteins produced in this way must be tested for safety and effectiveness. At this writing, an anti-clotting protein called anti-thrombin is now being tested in clinical trials.

Comparing this procedure to the use of bioreactor machines illustrates the fact that a generation of biochemical engineers failed to match the abilities of a tool for making proteins (the pig) that God had prepared. The mammary gland is optimized to maintain a high density of cells to deliver to them an ample supply of nutrients and to channel proteins that are produced in a form that can easily be isolated and purified. This procedure has proven to be successful and promises to provide a method for producing valuable therapeutic proteins at much lower cost.