from the column by treatment with a buffer that breaks the A:T hydrogen bonds.
Figure 2.10 Schematic representation of oligo(dT)-cellulose separation of polyadenylated mRNA from total cellular RNA.
To convert mRNA to double-stranded DNA for cloning, the enzyme reverse transcriptase, encoded by certain RNA viruses (retroviruses), is used to catalyze the synthesis of complementary DNA (cDNA) from an RNA template. If the sequence of the target mRNA is known, a short (∼20 nucleotides), single-stranded DNA molecule known as an oligonucleotide primer that is complementary to a sequence at the 3′ end of the target mRNA is synthesized (Fig. 2.11A). The primer is added to a sample of purified mRNA that is extracted from eukaryotic cells known to produce the mRNA of interest. This sample of course contains all of the different mRNAs that are produced by the cell; however, the primer will specifically base-pair with its complementary sequence on the target mRNA. Not only is the primer important for targeting a specific mRNA, but also it provides an available 3′ hydroxyl group to prime the synthesis of the first cDNA strand. In the presence of the four deoxyribonucleotides, reverse transcriptase incorporates a complementary nucleotide into the growing DNA strand as determined by the sequence of the template mRNA strand. To generate a double-stranded DNA molecule, the RNA:DNA (heteroduplex) molecules are treated with RNase H, which nicks the mRNA strands, thereby providing free 3′ hydroxyl groups for initiation of DNA synthesis by DNA polymerase I. As the synthesis of the second DNA strand progresses from the 3′ ends of the nicked mRNA fragments, the 5′ exonuclease activity of DNA polymerase I removes the ribonucleotides of the mRNA. After synthesis of the second DNA strand is completed, the ends of the cDNA molecules are blunted (end repaired and polished) with T4 DNA polymerase, which removes 3′ extensions and fills in from 3′ recessed ends. The double-stranded cDNA carrying only the exon sequences encoding the eukaryotic protein can be cloned directly into a suitable vector by blunt-end ligation. Alternatively, chemically synthesized short double-stranded DNA adaptors that contain a restriction endonuclease recognition sequence can be ligated to the ends of the cDNA molecules, and then digested with the restriction endonuclease prior to insertion into a vector via sticky-end ligation.
Figure 2.11 Synthesis of double-stranded cDNA using gene-specific primers (A) or oligo(dT) primers (B). A short oligonucleotide primer is added to a mixture of purified mRNA and anneals to a complementary sequence on the mRNA. Reverse transcriptase catalyzes the synthesis of a DNA strand from the primer using the mRNA as a template. To synthesize the second strand of DNA, the mRNA is nicked by RNase H, which creates initiation sites for E. coli DNA polymerase I. The 5′ exonuclease activity of DNA polymerase I removes RNA sequences that are encountered as DNA synthesis proceeds. The ends of the cDNA are blunted using T4 DNA polymerase prior to cloning.
When the sequence of the target mRNA intended for cloning is not known or when several target mRNAs in a single sample are of interest, cDNA can be generated from all of the mRNAs using an oligo(dT) primer rather than a gene-specific primer (Fig. 2.11B). The mixture of cDNAs, ideally representing all possible mRNA produced by the cell, is cloned into a vector to create a cDNA library that can be screened for the target sequence(s) (described below).
Recombinational Cloning
Recombinational cloning is a rapid and versatile system for cloning sequences without restriction endonuclease and ligation reactions. It is particularly useful when a large number of DNA fragments are to be cloned into one type of vector, for example, to introduce protein coding sequences into an expression vector for the production and purification of thousands of different proteins in parallel to facilitate the creation of a proteomic microarray (described later in this chapter). One method, known as Gateway cloning technology, exploits the mechanism used by bacteriophage λ to integrate viral DNA into the host bacterial genome during infection. Bacteriophage λ integrates into the E. coli chromosome at a specific sequence (25 bp) in the bacterial genome known as the attachment bacteria (attB) site. The bacteriophage genome has a corresponding attachment phage (attP) sequence (243 bp) that can recombine with the bacterial attB sequence with the help of the bacteriophage λ recombination protein integrase and an E. coli-encoded protein called integration host factor (Fig. 2.12A). Recombination between the attP and attB sequences results in insertion of the phage genome into the bacterial genome to create a prophage with attachment sites attL (100 bp) and attR (168 bp) at the left and right ends of the integrated bacteriophage λ DNA, respectively. For subsequent excision of the bacteriophage λ DNA from the bacterial chromosome, recombination between the attL and attR sites is mediated by integration host factor, integrase, and bacteriophage λ excisionase (Fig 2.12B). The recombination events occur at precise locations without either the loss or gain of nucleotides.
Figure 2.12 Integration (A) and excision (B) of bacteriophage λ into and from the E. coli genome via recombination between attachment (att) sites in the bacterial and bacteriophage DNA.
For recombinational cloning, a modified attB sequence is added to each end of the target DNA. The attB sequences are modified so that they will only recombine with specific attP sequences. For example, attB1 recombines only with attP1, and attB2 recombines with attP2. The target DNA with flanking attB1 and attB2 sequences is mixed with a vector (donor vector) that has attP1 and attP2 sites flanking a toxin gene that will be used for negative selection following transformation into a host cell (Fig. 2.13A). Integrase and integration host factor are added to the mixture of DNA molecules to catalyze in vitro recombination between the attB1 and attP1 sites and between the attB2 and attP2 sites. As a consequence of the two recombination events, the toxin gene sequence between the attP1 and attP2 sites on the donor vector is replaced by the target gene. The recombination events create new attachment sites flanking the target gene sequence (designated attL1 and attL2), and the plasmid with the attL1-target gene-attL2 sequence is referred to as an entry clone. The mixture of original and recombinant DNA molecules is transformed into E. coli, and cells that are transformed with donor vectors that have not undergone recombination retain the toxin gene and therefore do not survive. Host cells carrying the entry clone are positively selected by the presence of a selectable marker.
Figure 2.13 Recombinational cloning. (A) Recombination (thin vertical lines) between a target gene with flanking attachment sites (attB1 and attB2) and a donor vector with attP1 and attP2 sites on either side of a toxin gene results in an entry clone where the target gene is flanked by attL1 and attL2 sites. The selectable marker (SM1) enables selection of cells transformed with an entry clone. The protein encoded by the toxin gene kills cells transformed with nonrecombined donor vectors. The origin of replication of the donor vector is not shown. (B) Recombination between the entry clone with flanking attL1 and attL2 sites and a destination vector with attR1 and attR2 sites results in an expression clone with attB1 and attB2 sites flanking the target gene. The selectable marker (SM2) enables selection of transformed cells with an expression clone. The second plasmid, designated as a by-product, has the toxin gene flanked by attP1 and attP2 sites. Cells with an intact destination vector that did not undergo recombination or that retain the by-product plasmid are killed by the toxin. Transformed cells with an entry clone, which lacks the SM2 selectable marker, are selected against. The origins of replication and the sequences for expression of the target gene are
