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Ligation Reaction

DNA Ligation
The term recombinant DNA encapsulates the concept of recombining fragments of DNA from different sources into a new, and hopefully useful DNA molecule. Joining linear DNA fragments together with covalent bonds is called ligation. More specifically, DNA ligation involves creating a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. The enzyme used to ligate DNA fragments is T4 DNA ligase, which originates from the T4 bacteriophage. This enzyme will ligate DNA fragments having overhanging, cohesive endsthat are annealed together, as in the EcoRI example below this is equivalent to repairing "nicks" in duplex DNA. T4 DNA ligase will also ligate fragments with blunt ends, although higher concentrations of the enzyme are usually recommended for this purpose.

A ligation reaction requires three ingredients in addition to water:

Two or more fragments of DNA that have either blunt or compatible cohesive ("sticky") ends. A buffer which contains ATP. The buffer is usually provided or prepared as a 10X concentrate which, after dilution, yields an ATP concentration of roughly 0.25 to 1 mM. Most restriction enzyme buffers will work if supplemented with ATP. T4 DNA ligase. A typical reaction for inserting a fragment into a plasmid vector (subcloning) would utilize about 0.01 (sticky ends) to 1 (blunt ends) units of ligase.

The optimal incubation temperature for T4 DNA ligase is 16C and when very high efficiency ligation
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Ligation Reaction

is desired (e.g. making libraries) this temperature is recommended. However, ligase is active at a broad range of temperatures, and for routine purposes such as subcloning, convenience often dictates incubation time and temperature - ligations performed at 4C overnight or at room temperature for 30 minutes to a couple of hours usually work well. The figure to the right depicts the effects of T4 DNA ligase. DNA fragments generated by digestion of a plasmid with two restriction enzymes were incubated with different amounts of ligase for varying periods of time, then electrophoresed in 1% agarose. Note that even after 5 minutes with ligase, essentially all the fragments have been ligated to one another, and shifted to higher molecular weights. Click on the image for details. This simple test is sometimes useful to check a tube of ligase suspected of being "dead".
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Factors that Influence Enzyme Activity

Restriction

It is not uncommon to have difficulties in digesting DNA with restriction enzymes. At times, the DNA does not appear to cut at all and sometimes it cuts only partially. If the sequence is known, restriction sites can be predicted with accuracy, but in the lab, an enzyme may cut more often than it should or at the wrong sites. In some cases, these unexpected results point to a problem not related to technique - for example, the sequence you have may be incorrect, or a restriction map provided by a colleague could be in error. However, there are a number of commonly-encountered situtions that influence how well restriction enzymes cut, and it is important to be aware of these for troubleshooting.

Buffer Composition
Different restriction enzymes have differing preferences for ionic strength (salt concentration) and major cation (sodium or potassium). A battery of 3 to 4 different buffers will handle a large number of available enzymes, although there are a few that require a unique buffer environment. In all cases, a major function of the buffer is to maintain pH of the reaction (usually at 8.0). Additionally, some enzymes are more fussy about having their optimal buffer than other enzymes. Clearly, use of the wrong buffer can lead to poor cleavage rates.

Incubation Temperature
Most restriction enzymes cut best at 37C, but there are many exceptions. Enzymes isolated from thermophilic bacteria cut best at temperatures ranging from 50 to 65C. Some other enzymes have a very short half life at 37C and its recommended that they be
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Ligation Reaction

incubated at 25C.

Influence of DNA Methylation


Almost all strains of E. coli bacteria used for propagating cloned DNA contain two sitespecific DNA methylases:

Dam methylase adds a methyl group to the adenine in the sequence GATC, yielding a sequence symbolized as GmATC. Dcm methylase methylates the internal cytosine in CC(A/T)GG, generating the sequence CmC(A/T)GG.

The practical importance of this phenomenon is that a number of restriction endonucleases will not cleave methylated DNA. A few examples relative to Dam methylation should illustrate this concept: MboI and Sau3AI are isoschizomers that recognize and cleave the sequence GATC, which is precisely the sequence recognized by Dam methylase. Digestion of GmATC by MboI is completely inhibited, while digestion by Sau3AI is unaffected by methylation.

The recognition site for ClaI is ATCGAT, which is not a substrate for Dam methylase. However, if that sequence is followed by a C or preceeded by a G, a Dam recognition site is generated and cleavage by ClaI is inhibited. Thus, arandom sequence of DNA propagated in most strains of E. coli, half of the ClaI recognition sites will not cut.

The take-home message here is that if DNA unexpectedly does not cut or cuts only partially, check that the enzyme in question is not methylation-sensitive.

Star Activity
When DNA is digested with certain restriction enzymes under non-standard conditions, cleavage can occur at sites different from the normal recognition sequence - such aberrent cutting is called "star activity". An example of an enzyme that can exhibit star activity is EcoRI; in this case, cleavage can occur within a number of sequences that differ from the

Ligation Reaction

canonical GAATTC by a single base substitutions. So what constitutes non-standard conditions? Examples that may induce star activity include:

High pH (>8.0) or low ionic strength (e.g. if you forget to add the buffer) Glycerol concentrations > 5% (enzymes are usually sold as concentrates in 50% glycerol) Extremely high concentration of enzyme (>100 U/ug of DNA) Presence of organic solvents in the reaction (e.g. ethanol, DMSO)

Digestion with Multiple Enzymes


Digesting DNA with two enzymes is a commonplace task, and oftentimes the two enzymes have different buffer requirements. There are at least three ways to handle this situation:

Digest with both enzymes in the same buffer. In many cases, even those a given buffer is not optimal for an enzyme, you can still get quite good cleavage rates. Enzyme manufacturer catalogs usually contain a reference table recommended the best single buffer for conducting specific double digests. Cut with one enzyme, then alter the buffer composition and cut with the second enzyme. This usually applies to situations where one enzyme like a low salt buffer and the other a high salt buffer, in which case you can digest with the first enzyme for a time, add a calculated amount of concentrated NaCl and cut with the second enzyme. Change buffers between digestion with two enzymes. In some cases, two enzymes will have totally incompatible buffers. In that case, perform one digestion, recover the DNA (usually by precipitation) and resuspend in the buffer appropriate for the second enzyme.

Variability In Digestion of Different DNA Substrates


The efficiency with which a restriction enzyme cuts its recognition sequence at different locations in a piece of DNA can vary 10 to 50-fold. This is apparently due to influences of sequences bordering the recognition site, which perhaps can either enhance or inhibit enzyme binding or activity. A related situation is seen when restriction recognition sites are located at or very close to the ends of linear fragments of DNA. Most enzymes require a few bases on either side of their recognition site in order to bind and cleave. Many of the companies that sell enzymes provide a table in their catalog that presents "end requirements" for a variety of enzymes.

Ligation Reaction

E. coli DNA Polymerase I


The E. coli DNA polymerase I is a DNA-dependent DNA polymerase that possesses both 3' -> 5' and 5' -> 3' exonuclease activities. It is a single-chain protein with a mass of about 109,000 Da that requires magnesium as a cofactor. Each of its three enzymatic activities are encapsulated into distinct domains of the holoenzyme, such that proteolytic deletions can be generated that lack one or more of the activities. The so-called Klenow fragment is one such molecule that is widely used in recombinant DNA work. DNA polymerase I was used frequently in the early days of recombinant DNA technology for radiolabeling DNA and synthesizing cDNA. However, other enzymes have proven to be more effective for these purposes, including a proteolytic fragment of DNA polymerase I called Klenow fragment and T4 DNA polymerase. The holoenzyme DNA polymerase I is no longer frequently used. -------------------------------------------------------------------------------------------------------------

Large (Klenow) Fragment of E. coli DNA Polymerase I


The 5' -> 3' exonuclease activity of E. coli's DNA polymerase I makes it unsuitable for many applications. However, this pesky enzymatic activity can readily be removed from the holoenzyme. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5' -> 3' exonuclease activity, and a large piece called Klenow fragment. The large or Klenow fragment of DNA polymerase I has DNA polymerase and 3' -> 5' exonuclease activities, and is widely used in molecular biology.

Ligation Reaction

In addition to generating Klenow fragment by proteolysis, it can be expressed in bacteria from a truncated form of the DNA polymerase I gene. The enzyme you purchase is almost certainly produced in this manner. Klenow fragment is useful for several tasks: Synthesis of double-stranded DNA from single-stranded templates: The function of DNA polymerases is to synthesize complementary strands during DNA replication. Performing that task in the lab is integral to such processes as synthesizing the second strand DNA in cDNA cloning and generating radioactive probes for hybridization reactions. DNA polymerases require a primer to provide a free 3' hydroxyl group for initiation of synthesis. The primers used for most in vitro polymerization reactions are singlestranded DNAs, typically 6 to 20 bases in length, called oligonucleotides. The oligonucleotides must be complemenary to some section of template DNA. To use Klenow to synthesize a complementary strand of DNA, one simply mixes singlestranded template (usually denatured double-standed DNA), primers and the enzyme in the presence of an appropriate buffer (most restriction enzyme buffers work well). The reaction proceeds are depicted below:

One item of some significance in the above reaction is that as Klenow proceeds, it can displace primers downstream and continue synthesizing new DNA. Filling in recessed 3' ends of DNA fragments: A "fill-in" reaction is used to create blunt ends on fragments created by cleavage with restriction enzymes that leave 5' overhangs. This reaction is conceptually identical to the one described above, but with a

Ligation Reaction

huge primer and a very short segment of single-stranded template.

Digesting away protruding 3' overhangs: This is another method for producing blunt ends on DNA, in this with ends generated from restriction enzymes that cleave to produce 3' overhangs. The 3' -> 5' exonuclease activity of Klenow will digest away the protruding overhang. Removal of nucleotides from the 3' ends will continue, but, in the presence of nucleotides, the polymerase activity will balance the exonuclease activity, yielding blunt ends. This reaction is more efficienty conducted with T4 DNA polymerase, which has much more potent exonuclease activity.

Preparation of radioactive DNA probes: Examine each of the reactions depicted above. What if the nucleotides used were in the reaction were radioactive? That's correct - the radioactive nucletides would be incorporated into the DNA fragment. Klenow fragment is used frequently to prepare DNA that is labeled with radionuclides or other markers. In some situations, the 3' -> 5' exonuclease activity of Klenow fragment is either undesirable or not necessary. By introducing mutations in the gene that encodes Klenow, forms of the enzyme can be expressed that retain polymerase activity, but lack any exonuclease activity. These forms are the enzyme are usually called exo - Klenow fragment

Ligation Reaction

To perform ligation reaction using T4 DNA ligase.


Theory:Genetic engineering is a modern biotechnology technique which is used to modify the genetic makeup of an organism by adding new traits in to it and there by produce new variety of organisms. The desired trait or gene is cut from the donor gene and pasted to the carrying vector. Thus formed recombinant DNA is transferred into the host organism which has to be modified genetically. The process of Genetic engineering is made effective by the action of two enzymes namely, Restriction enzymes and ligases. Restriction enzymes, called molecular scissors are precisely cut the desired DNA and its carrying vector, at specific sites , whereas the ligase enzyme, called Molecular glue are pasted these DNA fragments into the carrying vector. The process of ligase enzyme for joining the two ends of DNA strands is called ligation. In this process, there occurs a synthesis of phosphodiester bond between the 3hydroxyl of one nucleotide and 5phosphate of another. In this situation we need to discuss about DNA and its Structure.

Structure of DNA:
DNA is made up of nucleotides which are composed of a nitrogenous base, sugar (ribose) and phosphate group. The structure thus formed is called as Double helix. The sugar and the phosphate group form the backbone of this helix. The sugar is attached to one of the four bases namely adenine, thymine, guanine and cytosine. The sugar is a pentose sugar called as the 2deoxyribose. This sugar is connected to the carbon atoms of the adjacent sugars by phosphate groups that make the phosphodiester bonds between the third carbon atoms of one sugar to the fifth carbon atom of the adjacent sugar. Ligating an insert DNA into a plasmid requires complementary ends between the DNA and the plasmid vector. Sticky ends are produced by cutting the DNA in a staggered manner within the recognition site and there by produce short Single stranded DNA. These ends have identical nucleotide sequence and are sticky because they can bind to complementary tails of other DNA fragments cut by the same

Ligation Reaction
restriction enzyme. Both are useful in molecular genetics for making recombinant DNA and proteins. Blunt ends are generated by cutting both DNA strands in the middle of the recognition sequence. DNA ligase helps to join together the complementary ends of insert DNA and plasmid DNA. Different parameters affect ligations such as the ratio of insert to vector, the quality and type of the DNA ends, the ligation temperature and the DNA concentration. Each of these factors is necessary for a successful ligation. The basic purpose in molecular cloning is the insertion of DNA fragment of interest (a segment of DNA) into a DNA molecule (called a vector) that has the capacity to replicate independently with in a host cell. The result is a recombinant molecule composed of the DNA insert joined to vector DNA sequences. Construction of these recombinant DNA molecules is dependent on the ability to covalently seal single stranded nicks in DNA. This process is accomplished both in vivo and in vitro by the enzyme DNA ligase.

The DNA fragments used to produce recombinant molecules are commonly generated by digestion with restriction endonucleases. Most of these enzymes cut their recognition sequences at staggered sites, giving overhanging or cohesive single-stranded tails. These are associate with each other by complementary base pairing. This paired complementary ends can be established permanently by DNA ligase treatment. Thus, two different fragments of DNA prepared by digestion with the same restriction endonuclease further joined to produce a recombinant DNA molecule.

Ligation Reaction

Principle:DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of making new viable DNA or plasmids. There are currently three methods for joining DNA fragments in vitro. The rst of these is DNA ligase that covalently joins the annealed cohesive ends produced by certain restriction enzymes. The second depends upon the ability of DNA ligase from phage T4-infected E. coli to catalyse the formation of phosphodiester bonds between sticky or blunt-ended fragments. The third utilizes the enzyme terminal deoxynucleotidyl transferase t o synthesize homopolymeric 3 singlestranded tails at the ends of fragments. The most commonly used is the T4 DNA ligase method.

E.coli and phage T4 encode an enzyme, DNA ligase, which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain. Although the reactions catalyzed by the enzymes of E. coli and T4-infected E. coli are very similar, they differ in their cofactor requirements. The T4 enzyme requires ATP, while the E. coli enzyme requires NAD+. In each case the cofactor is split and forms an enzymeAMP complex. The complex binds to the nick, which must expose a 5 phosphate and 3 OHgroup, and makes a covalent bond in the phosphodiester chain.

DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligases. During normal DNA replication, DNA ligase catalyzes the end-to-end joining (ligation) of short fragments of DNA, called Okazaki fragments. For purposes of DNA cloning, purified DNA ligase is given to covalently join the ends of a restriction fragment and vector DNA that have complementary ends. The vector DNA and restriction fragment are covalently ligated together through the 3 5 phosphodiester bonds of DNA. When termini created by a restriction endonuclease that creates cohesive ends associate, the nicks in the joints has few base pairs apart in opposite strands. DNA ligase can then repair these nicks to form an intact duplex.

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Ligation Reaction

T4 DNA Ligase
Bacteriophage T4 DNA ligase is a single polypeptide with a M.W of 68,000 Dalton requiring ATP as energy source. The maximal activity pH range is 7.5-8.0. The enzyme exhibits 40% of its activity at pH 6.9 and 65% at pH 8.3. The presence of Mg++ ion is required and the optimal concentration is 10mM.

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Ligation Reaction
T4 DNA ligase has the unique ability to join sticky and blunt ended fragments. Cohesive end ligation is carried out at 12C to 16C to maintain a good balance between annealing of ends and activity of the enzyme. If reaction is set at higher temperatures annealing of the ends become difficult, while lower temperatures diminishes the ligase activity. All T4 DNA ligase is inactivated by heating at 65C for 10 minutes. Beside of these ligating complementary sticky ends, T4 ligase can ligate any two blunt DNA ends. Lack of cohesive termini makes blunt end ligation more complex and significantly slower. Since annealing of ends is not a factor, the reaction is done at 24C. However, 10 - 100 times more enzyme is required to achieve similar ligation efficiency as that of cohesive end ligation. The enzyme has involved in the catalysis of the joining of RNA to either a RNA or DNA strand in a duplex molecule but this will not involved in the joining of single stranded nucleic acids.

Applications:
Ligation of cohesive or blunt-ended DNA fragments for cloning Sealing nicks in double-stranded DNA Ligation of synthetic linkers to blunt-ended DNA Three main components of a ligation reaction are:1) Two or more fragments of DNA that have compatible ends. 2) A buffer that has 0.25-1mM ATP to provide the necessary energy for the reaction. 3) The T4 DNA ligase. The two components of the DNA in the ligation reaction (vector and insert) should be equimolar and around 100g/ml. Usually, one wants to ligate an insert DNA molecule into a plasmid, ready for bacterial transformation. Typically, DNA and plasmid vector are separately cleaved to get complementary ends, then both are added to a ligation reaction to be circularised by DNA ligase. If the ratio of plasmid backbone to insert DNA is too high then excess 'empty' mono and polymeric plasmids will be generated. If the ratio is too low results in an excess of linear and circular homo- and heteropolymers.The ideal ratios for ligating insert to vector for sticky end ligations ranges between 1:1 and 3:1, where as for blunt ended ligations, the insert to vector ratio should be at least 10:1.

Calculating Insert Amount:-

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