CLONES
Basic Plasmid Cloning
Restriction enzymes are used to cleave the vector, and cleave a compatible fragments out of the insert. DNA ligase is used to join insert to vector. The mixture is transformed into E. coli, and then the bacteria are cloned. Each clone will have only one of the possible ligation products, because of 'incompatibility' (meaning that if multiple plasmids of the same kind enter a bacterium, all but one of them gets kicked out).
Modern cloning vectors have engineered multiple cloning sites. This is the map of pUC19, from the New England BioLabs catalog:
For a result with the minimal amount of trouble, one should gel purify both the cleaved vector and the cleaved insert before ligating. This removes extraneous DNA species that may interfere (the most troublesome of which is residual uncleaved vector). It also allows verification that a fragment of the expected size has in fact been produced, and allows crude quantitation of vector and insert. Vector and insert are usually ligated in ~ 1:1 molar ratios. Excessive amounts of either or both can lead to concatemerization instead of circle formation. Concatemers do not produce colonies on transformation. Interfering enzyme activities can usually be removed by a heat treatment.
Type II restriction enzymes leave ends that may be 5' overhanging, 3' overhanging, or blunt. In all cases each end is left with a 3' OH and a 5' phosphate. All blunt ends, and any complementary overhanging ends may be religated with T4 DNA ligase, as long as at least one 5' phosphate is present. A common cloning strategy is to dephosphorylate the vector. This prevents ligation of vector ends to each other, but does not inhibit ligation of vector to insert. Dephosphorylation has no impact on whether a restriction site is restored after ligation.
In the tables below G^AATTC means that the end will be:
-----G 3'
-----CTTAA 5'
GTGCA^C means that the end will be:
-----GTGCA 3'
-----C 5'
Compatible ends
Isoschizomers - Enzymes that recognize the same sequence. Isoschizomers may not leave the same kind of end (and also may not be affected the same by methylation).
Type II enzymes cleaving different recognition sites may leave complementary (compatible) ends. Clones that are made by joining compatible ends may not regenerate either of the restriction sites, or they may generate some other site. For example an AccI cut insert joined to a ClaI cut vector will create a junction recognized as a TaqI site.
Examples of compatibility groups:
AccI GT^CGAC BamHI G^GATCC HgiAI GTGCA^C
ClaI AT^CGAT BclI T^GATCA NsiI ATGCA^T
FspII TT^CGAA MboI ^GATC PstI CTGCA^G
TaqI T^CGA BglII A^GATCT
All blunt ends are compatible:
HaeIII GG^CC
AluI AG^CT
SmaI CCC^GGG
HincII GTY^RAC; R is A or G; Y is C or T
Some other commonly used restriction enzymes that are incompatible with other ends are listed below. Notice that for some cases ends produced by the same enzyme are not necessarily compatible (eg. HinfI).
EcoRI G^AATTC
HindIII A^AGCTT
SacI GAGCT^C
HinfI G^ANTC; N is A,T,G, or C
DdeI C^TNAG
BglI GCCN4^NGGC
Verifying a construct by restriction endonuclease cleavage:
Most typically restriction digests are assayed by electrophoresis on a small gel of 0.7 to 1% agarose. This technique is fast and simple. Although polyacrylamide gels have higher resolution for fragments below 500 bp, most people try to get by with higher percentage agarose, or certain brands of agarose engineered for resolution of smaller fragments. Capillary gel electrophoresis is also possible, but the equipment available within the department is configured for fluorescently labeled fragments, and is only applicable to special applications.
Visualization: Although other staining agents are available, most commonly the DNA is visualized by staining in 0.5 ug/ml ethidium bromide, which can either be soaked into the gel or placed in the gel during its original formation. Visualization requires illumination with a UV light. A wave length of 265 nm is most effective, but also does more damage to the DNA and is a safety concern. Illumination at 300 nm is common, particularly if the DNA is to be recovered from the gel for cloning. For imaging the gel may be either illuminated from below or above. The investigator should never stare into a gel laying on top of a UV light source, at least not without a UV face shield. There are a number of high tech imaging devices ranging from specialized gel imagers to home-made CCD cameras mounted on laboratory computers. The Biochemistry Dept. has a departmental imager in 550C. Contact Sandy Mathis to arrange the required brief training if you wish to use it. Sensitivity typically allows visualization down to 5-10 ng in a band. One should always record an image of every gel.
The experiment depicted above is suboptimal for a number of reasons. Can you see why? There should be more markers, and there should be markers extending below the lowest experimental band of interest and markers better defining the onset of curvature. The gel could have been run longer to make better use of its revolving power.
· Understanding circular DNA
Plasmid DNA as isolated from cells is a mixture of forms: monomer covalently closed circle (ccc) (supercoiled), dimer and higher forms of ccc, and nicked circles (alias relaxed circles). The supercoils generally run as if they were about half the size of the corresponding linear, although the relationship between ccc and linear migration is dependent on gel conditions. The sizing curve for circles remains linear higher up on the gel.
On storage, ccc DNA converts slowly to the nicked circle (not linear) form. As soon as one cleaves with a restriction enzyme, the pattern simplifies to the pattern of bands you would expect from the map of the plasmid.
JOINING INCOMPATIBLE ENDS
Incompatible ends may be polished to blunt ends to permit ligation either by cleaving off the overhang, or by filling in the overhang.
Alternatively, a synthetic linker may be used. The following linker is used to join blunt ended fragments to an EcoRI site:
5'-pCCGGAATTCCGG-3'
Note that the linker is self complementary. When annealed to itself and ligated in excess to blunt ended fragments, it creates an array of RI sites at each end:
RRRRRRR RRRRRRR RRR
||||||| frag. ||||||| frag. |||
------------------------------------
Recleavage with RI leaves a fragment with RI ends. Note that you might have to first methylate the fragment with EcoRI methylase to protect any internal EcoRI sites during the recleavage. Alternatively, you could create the overhang directly with two separate oligos, thus avoiding the recleavage:
5'-AATTCCCGGG-3'
GGGCCCp-5'
Note that by leaving the phosphate off of the upper strand, you preclude joining of linker RI ends to each other. The phosphates for joining to the vector will have to be provided by the vector ends.
Public repositories of cDNA clones and sequence information.
Large projects are underway to obtain and array cDNA clones from human, mouse, rat, and zebrafish, etc; to add their sequences to the EST database; and to make the clones physically available. NIH's project is called Mammalian Gene Collection (http://mgc.nci.nih.gov/). If you identify a clone you want by a database search, you can obtain the cDNA clone from an associated supplier called the I.M.A.G.E. Consortium. See: http://image.hudsonalpha.org/ (Bonaldo, Lennon and Soares, Genome Res., 1996, 791-806). An index of other projects attempting to compile full length cDNA clones for a variety of genomes is found at http://www.ncbi.nlm.nih.gov/genome/flcdna. A number of commercial enterprises sell clones from their own collection: http://www.genecopoeia.com/, http://www.origene.com/cdna/, and http://clones.invitrogen.com.
NCBI has a help page for finding cDNA clones through its database system: http://www.ncbi.nlm.nih.gov/genome/clone/finding_cdna.shtml.
Map browsers for various genomes exist to enable correlating cDNA structure with splicing patterns of various genomes:
· http://www.ncbi.nlm.nih.gov/Genomes/ - index of NCBI's map viewers.
· http://genome.ucsc.edu/cgi-bin/hgGateway?org=human - Gatway to UCSC's genome browsers.
· http://www.ensembl.org/index.html - ensemble genomes
· http://img.jgi.doe.gov/cgi-bin/pub/main.cgi - D.O.E.'s Integrated Microbial Genomes.
PCR Amplification from mRNA to recover or extend a cDNA.
If one conducts first strand synthesis with reverse transcriptase, it is possible to add PCR primers and complete the synthesis of a cDNA, as well as amplifying it, in the PCR machine. This is sometimes called RT PCR (although the designation 'RT' is also often used to mean 'real time' instead of 'reverse transcription'). Both primers could be specific for a particular gene, or one specific primer could be used with oligo dT.
The following figure shows amplification between a gene specific primer (GS1) and the poly(A) tail which has incorporated two improvements over the basic technique. 1) The reverse transcription is conducted with oligo dT with a 5' extension. The PCR primer matches the 5' extension. This avoids trying to amplify with oligo dT itself, which would force badly balanced TM's between the two primers. 2) The first population of cDNAs produced are re-amplified with a second (nested) gene-specific primer. This restores the specificity of a two primer amplification.
A similar procedure can be used to extend towards the 5' end.
This procedure has been named RACE (Rapid Amplification of cDNA Ends).
Ref: Frohman et al., PNAS 85, 8998-9002 (1988) Current Protocols in Molecular Biology (Ausubel et al.) has a more thorough accounting of this an most basic cloning procedures.
Directly Cloning PCR Products
Remember that PCR primers as delivered from an oligonucleotide synthesis facility do not have 5' phosphates. Hence a mixture of blunt ended PCR products should not be able to ligate to one another. They can join to a blunt ended vector, if the vector has 5' phosphates. However, the common practice of dephosphorylating the vector to prevent circularization without an insert would also block ligation to the insert. In this case, one would have to phosphorylate either the PCR primers before amplification, or the PCR product after amplification. Since single stranded ends are more reliably phosphorylated, it would be best to do it at the primer level. The classic way to measure efficiency of a phosphorylation reaction is to use g[32]P-ATP, and then measure the specific activity of the product. Since PCR products typically have 3' overhangs (see below), direct cloning is inefficient unless one first 'polishes' the ends by reaction with a DNA polymerase with 3' -> 5' exonuclease activity. The most commonly used enzymes are T4 DNA polymerase or Pfu DNA polymerase (see below).
TA cloning
During PCR, the polymerase tends to add an extra untemplated base to the 3' ends of each synthesized fragment. Most commonly, an A is added, essentially making a one base sticky end. This arrangement can be used for highly efficient cloning into a vector cleaved so as to leave a 3' T overhang. The most popular systems are vended by InVitrogen, and are based on "Topo Cloning". In this procedure, the vector was opened by a topoisomerase instead of a restriction enzyme. The topoisomerase cleaves to leave the 3' T overhang, but also remains covalently attached to the vector. When provided a PCR product with an A overhang, the topoisomerase will reverse its reaction and join the PCR product to the vector. No ligase in involved, and phosphorylation of the 5' ends of the PCR product is unnecessary.
TOPO® TA Cloning® of Taq-amplified DNA (From the InVitrogen web site).
The topo cloning procedure is highly efficient. It does have the drawback that the vector is expensive and loses efficiency during on storage. It also has the drawback that one is limited as to the choice of vectors and vector sites into which to insert. One could engineer any blunt end restriction site in a vector for TA cloning by cleaving the vector and then adding the T overhang by exposing to Taq polymerase + dTTP. In this case the joining would require ligase, and one would have to pay attention to 5' phosphates.
Different polymerases differ in their propensity to add the overhang, and in their preference of which base to add [Hu, DNA Cell Biol. 12:763-70(1993)]. Furthermore, the identity of the last templated base on the 3' end of the PCR product influences the efficiency of adding the overhang, and the identity of the base to be added. For example, Taq polymerase most efficiently adds an A if the last templated base is a C. Hence, I recommend a practice of putting a G on the 5' end of PCR primers if possible. Even if TA cloning is not part of the intended strategy, it helps to have it as a fallback option if the intended strategy fails to produce clones.
Pfu polymerase (best known for conducting PCR with higher fidelity than Taq polymerase) has a 3' exonuclease that precludes leaving an overhang. Hence Pfu polymerase would be a good choice if blunt end ligation was planned. However, Pfu products could be given the overhang by exposure to Taq polymerase plus dATP after the amplification, and Taq products can be polished to blunt ends by exposure to Pfu polymerase after amplification. Finally, the addition of the overhang by Taq polymerase is promoted by adding an extra 10 minute extension after the last cycle. If you use Taq and don't want the overhang, You should remove this extension from the procedure and also keep the other extension times as short as is practical.
Cloning PCR products with Restriction sites.
Alternatively, one can add a restriction site to the back of the PCR primers, and then cleave the product to expose the cohesive ends. It is best to gel purify the PCR product before attempting the cleavage, because the DNA polymerase will otherwise modify the ends exposed by the cleavage. PCR primers may also interfere with some restriction enzymes. After cleavage the fragments can then be ligated into a restriction enzyme-cleaved vector in the ordinary way. It helps to gel purify the fragments again after cleavage, since the little ends cleaved away will religate to both vector and insert and inhibit production of the desired product.
Six extra bases should be added behind each restriction site, so that the enzyme is not trying to cut too close to the end of the fragment.
Troubleshooting:
A common problem with this strategy is that either not enough restriction enzyme is used to cut off, or too much is used and the ends are damaged making them unligatable.
· Not enough enzyme: Restriction enzymes generally come with activity labeled in units similar to 10U/ul, meaning 1 ul of enzyme will cleave all EcoRI sites in 10 ug of some test DNA in 1 hour under suitable reaction conditions. Typically one uses a 10 fold excess to assure cleavage, given that some sites are slower to cut than others, and the enzyme may have lost some potency in storage. But the Unit = 1 ug/hour definition assumes some particular density of EcoRI sites. If the test molecule was typical of ordinary DNA sequence, it will have about 1 EcoRI site/2000 bp ( 1/4^6). For a 400 bp PCR product with an EcoRI site on each end, the concentration of EcoRI sites is 10x higher than the test molecule the enzyme was assayed against. It will be necessary to up the amount of enzyme used (or the length of the reaction) by 10 fold to get comparable cleavage efficiency. Another problem is that sites near the end of a DNA molecule tend to exhibit suboptimal cleavage.