cDNA is a DNA copy synthesized from mRNA. The enzyme used is reverse transcriptase an RNA-dependent DNA polymerase isolated from a retrovirus (AMV or MMLV). As with other polymerases a short double-stranded sequence is needed at the 3' end of the mRNA which acts as a start point for the polymerase. This is provided by the poly(A) tail found at the 3' end of most eukaryotic mRNAs to which a short complementary synthetic oligonucleotide (oligo dT primer) is hybridized (polyT-polyA hybrid). Together with all 4 deoxynucleotide triphosphates, magnesium ions and at neutral pH, the reverse transcriptase synthesises a complementary DNA on the mRNA template. The progress of synthesis can be monitored by incorporation of P-32 radioactive nucleotides.
Each mRNA molecule in the mixture with a poly(A) tail can be a template and will produce a cDNA in the form of a single stranded molecule bound to the mRNA (cDNA:mRNA hybrid). The cDNA requires to be converted into a double stranded DNA before it can be manipulated and cloned. This is carried out using another DNA polymerase - DNA Pol I (Klenow fragment). This is the large fragment (75kDa) of DNA polymerase I following its proteolysis with subtilisin; the resulting enzyme has 5'-3' polymerase activity and 3'-5' exonuclease activity but has lost the 5'-3' exonuclease activity associated with the whole enzyme. Commercial sources of the Klenow enzyme use a truncated pol A gene cloned and expressed in E.coli. Klenow polymerase is used to avoid degradation of the newly synthesised cDNAs. To produce the template for the polymerase the mRNA must be removed from the ss cDNA:mRNA hybrid. This is achieved either by boiling or by alkaline treatment (see lecture notes on the properties of nucleic acids). The resulting ss cDNA is used as the template to produce the second DNA strand. As with other polymerases a double stranded primer sequence is needed and this is fortuitously provided during the reverse transcriptase synthesis which produces a short complementary tail at the 5' end of the cDNA. This tail loops back onto the ss cDNA template ( the so-called hairpin loop ) and provides the primer for the polymerase to start the synthesis of the new DNA strand producing a double stranded cDNA (ds cDNA). A consequence of this method of cDNA synthesis is that the two complementary cDNA strands are covalently joined through the hairpin loop ie. the ds cDNA is essentially a single molecule ( shown by electrophoresis on denaturing gels ). The hairpin loop is removed by use of a single strand specific nuclease ( S1 nuclease from Aspergillus oryzae ).
Another strategy for cDNA synthesis employs a ribonuclease ( RNase H ) which recognises the RNA component of a DNA:RNA hybrid and cleaves the RNA at a number of non-specific sites leaving short oligoribonucleotides attached to the cDNA. These serve as primers for the polymerase to synthesise the second strand cDNA; DNA Pol I (not Klenow) is used since the 5'-3' exonuclease activity is needed to remove RNA in front of the enzyme. The newly synthesised strands of cDNA are joined by ligation using T4 ligase enzyme (+ATP).
Since the cDNA's resulting from either of the first two methods have no specific ends (ie restriction sites or sticky ends) they sometimes need to be manipulated for efficient joining to a vector DNA molecule. If the cDNA's are blunt-ended they may be cloned directly into a vector which has been cut with any blunt end cutting restriction enzyme such as Sma I. Blunt-end ligations are usually much less efficient than sticky end ligations and so it is usual to provide cDNA's with cohesive ends.
Synthetic cohesive ends can be attached (ligated) to cDNA's with blunt ends. Commonly an oligonucleotide containing a specific restriction site (usually for an enzyme producing a sticky or cohesive end) is made; since most restriction sites are palindromic the synthetic oligonucleotide immediately hybridises to itself ( becomes double-stranded ). These oligos or 'linkers' are then attached to the ends of the cDNA's using T4 ligase (+ATP); chemically synthesised DNA does not have 5' phosphate groups (5' hydroxyl groups only) essential for this ligation and so oligonucleotides are treated with T4 polynucleotide kinase (+ATP) which phosphorylates the 5' ends. Following ligation to the cDNA's they are then treated with the corresponding restriction enzyme to produce cDNA's with cohesive termini suitable for efficient ligation to a vector with compatible cohesive ends. Ligation of synthetic ends to DNA is efficient because high concentrations of oligonucleotides can be used.
A drawback when using the oligonucleotide linker approach is that any cDNA's containing a restriction site for the same enzyme whose site is incorporated into the oligonucleotide will be cleaved internally when the ends are cleaved, to produce two truncated cDNA molecules. One way to avoid this is to protect any internal restriction sites in the cDNA's by pre-treatment with the methylase enzyme (also known as a methyltransferase) specific for the restriction site being used in the oligos. For example if Hind III oligos are being used the cDNA's can be methylated using Hind III methylase and S-adenosyl methionine (methyl group donor) which protects any internal Hind III sites from subsequent restriction with Hind III. After protection the methylase enzyme is removed and the oligos attached as before. ( For further information see references to host-specific restriction-modification systems in bacteria ).
Ligation will join any compatible DNA ends irrespective of the DNA molecule to which they are attached. In a conventional cloning operation using a plasmid vector with a unique restriction site the majority of the ligation reaction rejoins the restricted plasmid back to itself (self-ligation). This is a consequence of the relatively high concentration of compatible ends - the two compatible sequences at either end of the same molecule! One trick which is often used to prevent an unwanted ligation is to remove the 5' phosphate group from the ends - ligation requires a 5' phosphate group and a 3' hydroxyl group on the ends of the compatible DNA fragments. The enzyme used for this is alkaline phosphatase from bacterial sources or calf intestines. Once treated DNA fragments (eg plasmid) cannot ligate back together. In cDNA cloning this means that ligation can only take place between the plasmid and a cDNA - only a recombinant plasmid is produced (ie one containing an insert).
Another technique used to efficiently clone cDNA's is to produce cohesive ends on cDNA's and plasmid exploiting the activity of terminal transferase, a template independent DNA polymerase. This enzyme from calf thymus catalyses the addition of 'homopolymer tails' to the 3' ends of the DNA; in the presence of a single base nucleotide (eg dCTP) the enzyme builds up a string of C's ( poly C tail ) at both ends of the DNA's. It will use either blunt ended DNA or 3' protruding ends produced by restriction enzymes such as Pst I. Choosing compatible homopolymer tails on cDNA's ( eg. poly C ) and vector ( eg. poly G ) allows these ends to anneal (hybridise) together so inserting the fragment into the vector. Using this system there is no requirement to ligate since the inserted fragment is stably maintained until the transformed E.coli host enzymes repair any gaps and covalently seal the plasmid.
The development of the Polymerase Chain Reaction (PCR) using the thermostable DNA polymerase Taq polymerase has seen several applications develop for the production and cloning of cDNA's particularly in situations where only very small quantities of mRNA is available (small amounts of tissue; low abundance of mRNA). The example shown in the three figures below is only one of many techniques which combine the activities of reverse transcriptase and Taq polymerase in what has become known as RT-PCR. The objective in most of these approaches is to provide sequences at both ends of the cDNA's to which complementary primers can be hybridised to allow amplification of the sequence between. In the strategy shown below the poly A tail of the mRNA is used initially (as in the traditional cDNA synthesis) to provide the start point for the reverse transcriptase using oligo dT as the primer. Note the modified oligo dT primer contains additionally a restriction site sequence ( Bam HI ). The initial synthesis of cDNA is otherwise exactly the same as in the traditional method. Following the synthesis of the first strand of cDNA and removal of the mRNA ( see earlier), the 3' end of the cDNA is homopolymer tailed using dGTP and terminal transferase - this produces a poly G tail at the 3' end. The poly G end can then be used in a similar fashion to the poly A end in the original mRNA.
A second primer - oligo dC can now be used to provide a start point for the DNA polymerase to synthesise the second starnd of cDNA. Note i) that the oligo dC primer also has a restriction site ( Hind III ) incorporated at its end and ii) there is now a primer site at both ends of the cDNA ( the requirement for PCR amplification ). In the presence of the oligo dC primer, dNTP's and Taq polymerase the second strand of cDNA is completed. Note that the synthesis proceeds through the poly A region and completes the retriction site at the 3' end ( Bam HI ). Thermal cycling in the presence of both primers ( oligo dT and oligo dC ) Taq polymerase will amplify the sequences between the two primers in a typical PCR reaction - note that the second restriction site at the 5' end is also completed so that all amplified cDNA's will contain a Hind III site at the 5' end and a Bam HI site at the 3' end. Note also because of these restriction sites that this particular strategy allows directional cloning of cDNA's - useful if an expression library is planned for screening with antibodies. After amplification the final step is the double restriction of the cDNA's with Hind III and Bam HI and ligation into a similarly cut plasmid vector followed by transformation. Note that a plasmid vector cut with two different restriction enzymes will not have compatible ends and cannot therefore be rejoined to itself ( self-ligated ); only if a fragment with suitably compatible ends ( ie a cDNA ) is inserted will a (recombinant) plasmid be formed.
For further information please consult items in the recommended reading lists or contact Dr R.Croy.
This page maintained by Ron Croy, 20th April 1998
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