178 POLYMERASE CHAIN REACTION 4 AAA-3′ 5′ 5′ 3′ mRNA Primer 1 AAA-3′ 5′ mRNA 5′ 3′ DNA 5′ 3′ 5′ 3′ 5′ 3′ 5′ 5′ Primer 1 3′ 3′ Primer 2 3′ 5′ 5′ 3′ 3′ 5′ Primer 2 RT PCR cycle 1 PCR cycle 2 RT Ta q Amplified product Figure 4.12. RT–PCR. The enzyme reverse transcriptase uses a complementary oligonu- cleotide to prime DNA synthesis from an RNA molecule. The single strand of DNA produced is then used as a template for the synthesis of a second DNA strand, and then for amplification by PCR devised as a method of RNA amplification and quantitation after its conversion to DNA. RT–PCR can be used for cloning, cDNA library construction and probe synthesis. The technique consists of two parts (Figure 4.12) – the syn- thesis of DNA from RNA by reverse transcription (RT) and the subsequent amplification of a specific DNA molecule by polymerase chain reaction (PCR). The RT reaction uses an RNA template (typically either total RNA or polyA + RNA), a primer (random or oligo dT primers), dNTPs, buffer and a reverse transcriptase enzyme (which we will discuss more in Chapter 5) to generate a single-stranded DNA molecule complementary to the RNA (cDNA). The cDNA then serves as a template in the PCR reaction. During the first cycle of 4.9 REAL-TIME PCR 179 PCR, the single DNA strand is made double stranded through the binding of another, complementary, primer and the action of Taq DNA polymerase. Like other methods of mRNA analysis, such as northern blots and nuclease protection assays, RT–PCR can be used to quantify the amount of mRNA that was contained in the original sample. This type of analysis is particularly important for monitoring changes in gene expression. However, because PCR amplification is exponential, small sample-to-sample concentration and loading differences are amplified as well. Even large differences in target concentration (100-fold or more) may produce the same intensity of band after 25 or 30 PCR cycles. Therefore, RT–PCR requires careful optimization when used for quan- titative mRNA analysis. Quantitation usually takes one of two forms – relative or absolute. • Relative quantitation compares transcript abundance across multiple sam- ples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene specific signal to the internal control signal. This yields a corrected relative value for the gene specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples. • Absolute quantitation, using competitive RT–PCR, measures the absolute amount (e.g. 5.3 × 10 5 copies) of a specific mRNA sequence within a sample. Dilutions of a synthetic RNA (containing identical primer binding sites, but slightly shorter than the target RNA) are added to the sample and are co-amplified with the target. The PCR product from the endogenous transcript is then compared with the concentration curve created by the synthetic competitor RNA. 4.9 Real-time PCR Quantitative real-time RT–PCR combines the best attributes of both relative and competitive RT–PCR in that it is accurate, precise, high throughput and relatively easy to perform. Real-time PCR automates the otherwise laborious process of relative RT–PCR by quantitating reaction products for each sample in every cycle. Real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, whose signal increases in direct proportion to the amount of PCR product in a reaction. In the simplest form, the reporter is the double-strand DNA-specific dye SYBR  Green (Wittwer et al., 1997). SYBR Green binds double-stranded DNA, probably in the minor groove, and, upon excitation, emits light. Thus, if the dye is included in a PCR reaction, as a 180 POLYMERASE CHAIN REACTION 4 3′ 5′ 3′ 5′ 3′ 5′ 3′ 5′ 3′ 5′ Primer 1 5′ 3′ QR Primer 2 Probe 5′3′ 3′ 5′ 3′ 5′ 5′ 5′ Q R 3′ 3′ 5′ 3′ 5′ 5′ 5′ Q R 3′ 3′ 5′ 3′ 5′ 5′ 5′ Q R Displacement Cleavage Completed synthesis Primer binding Figure 4.13. TaqMan  real-time PCR quantification. Three primers are used during the PCR process – two of these (primers 1 and 2) dictate the beginning of DNA replication on each DNA strand, and the third (the probe) binds to one strand in between. The probe contains two modified bases – a fluorescent reporter (R) at its 5  -end and a fluorescence quencher (Q) at its 3  -end. As DNA replication proceeds, the extended product from primer 1 displaces the 5  -end of the probe and the exonuclease activity of the polymerase cleaves the fluorescent reporter from the probe. The separation of the reporter from the quencher allows it to fluoresce. The amount of fluorescence is proportional to the amount of PCR product being made and is measured during each PCR cycle PCR product accumulates the fluorescence increases. The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer dimers and other non-specific reaction products, which can 4.10 APPLICATIONS OF PCR 181 result in an over-estimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles. The alternative method for quantifying PCR products is TaqMan  ,which relies on fluorescence resonance energy transfer (FRET) of hybridization probes for quantitation (Figure 4.13). TaqMan probes are oligonucleotides that con- tain a fluorescent reporter dye, typically attached to the 5  base, and a quenching dye, typically attached to the 3  base. The probe is designed to hybridize to an internal region of a PCR product. When irradiated, the excited reporter dye transfers energy to the nearby quenching dye molecule rather than fluorescing, resulting in a non-fluorescent substrate. During PCR, when the polymerase replicates a template on which a probe is bound, the 5  -3  exonuclease activ- ity of the polymerase cleaves the probe (Holland et al., 1991). This separates the fluorescent and quenching dyes and FRET no longer occurs. Fluorescence increases in each PCR cycle, proportional to the rate of probe cleavage, and is measured in a modified thermocycler. Real-time PCR is a powerful quantitative tool, but the cost of reagents and equipment is much higher than that of standard PCR reactions. 4.10 Applications of PCR The polymerase chain reaction has revolutionized molecular biology by allow- ing the amplification and characterization of minute amounts of nucleic acids. As well as being of use to basic scientists, this technique is of immense impor- tance in medicine for the identification of mutations within small amounts of human DNA, and to pathologists, who routinely need to detect and character- ize small amounts of infectious micro-organisms. A detailed description of the many and varied uses to which PCR has been applied is beyond the scope of this text, but a few of the major applications of PCR are listed below: • molecular cloning • DNA sequencing • archaeology • forensics • amplification of unknown sequences • clinical pathology • genetic diagnosis • characterizing unknown mutations 182 POLYMERASE CHAIN REACTION 4 • fingerprinting/population analysis • genome analysis • quantitative PCR of RNA or DNA. We will touch on some of these topics in later chapters but, again, interested readers are directed toward more dedicated literature (McPherson and Møller, 2000; Innis, Gelfand and Sninsky, 1999). 5 Cloning a gene Key concepts  DNA libraries are pools of recombinant DNA molecules  Genomic libraries contain fragments of all DNA sequences present in the genome  cDNA libraries contain DNA copies of mRNA and are tissue and developmental stage specific. Their formation is dependent on an RNA-dependent DNA polymerase enzyme, reverse transcriptase  PCR based libraries negate the requirement for cloned DNA frag- ments and can undergo subtraction to isolate genes that are differentially expressed Genomes contain an enormous amount of DNA (Table 5.1). Consequently, each gene contained within a genome represents only a tiny fraction of the genome size itself. All traditional DNA cloning strategies are composed of four parts: the generation of foreign DNA fragments, the insertion of foreign DNA into a vector, the transformation of the recombinant DNA molecule into a host cell in which it can replicate and a method of selecting or screening clones to identify those that contain the particular recombinant we are interested in. In this chapter we will address some of the particular problems and issues with the first two of these steps in the formation of DNA libraries. A DNA library is simply a collection of DNA fragments. There are several different types of library that we will consider here. DNA fragment libraries are designated as being either a genomic DNA library or a cDNA library. Most traditional methods of library construction involve the physical cloning of various DNA fragments into a suitable vector. However, as we will see later, DNA fragments that are not cloned (e.g. those derived Analysis of Genes and Genomes Richard J. Reece  2004 John Wiley & Sons, Ltd ISBNs: 0-470-84379-9 (HB); 0-470-84380-2 (PB) 184 CLONING A GENE 5 Table 5.1. Some fully sequenced genomes Organism Genome size (bp) Number of chromosomes (haploid where appropriate) Number of protein-coding genes Reference Methanococcus jannschii (archaebacteria) 1.7 × 10 6 1 1 750 (Bult et al., 1996) Haemophilus influenzae (virus) 1.8 × 10 6 1 1 850 (Fleischmann et al ., 1995) Bacillus subtilis (bacterium) 4.2 × 10 6 1 4 100 (Kunst et al., 1997) Escherichia coli (bacterium) 4.6 × 10 6 1 4 288 (Blattner et al., 1997) Streptomyces coelicolor (actinomycete) 8.7 × 10 6 1 7 825 (Bentley et al., 2002) Schizosaccharomyces pombe (yeast) 1 .4 × 10 7 3 4 824 (Wood et al., 2002) Saccharomyces cerevisiae (yeast) 1.4 × 10 7 16 6 184 (Goffeau et al., 1997) Caenorhabditis elegans (worm) 0.97 × 10 7 6 19 000 (The C. elegans Sequencing Consortium, 1998) Drosophila melanogaster (fruit fly) 1 .2 × 10 8 4 13 500 (Adams et al., 2000) Anopheles gambiae (malaria mosquito) 2.7 × 10 8 5 13 700 (Holt et al., 2002) Arabidopsis thaliana (plant) 1.2 × 10 8 5 25 498 (The Arabidopsis Genome Initiative, 2000) Oryza sativa (rice) 4 .3 × 10 8 12 ∼50 000 (Yu et al., 2002; Goff et al., 2002) Mus musculus (mouse) 2.5 × 10 9 21 ∼22 000 (Mouse Genome Sequencing Consortium, 2002) Homo sapiens (human) 2.9 × 10 9 23 ∼32 000 (Lander et al., 2001; Venter et al., 2001) 5.1 GENOMIC LIBRARIES 185 from PCR products) are becoming increasingly important in genetic engineering experiments. A genomic DNA library should contain representative copies of all the genetic material of an individual organism. Libraries such as this are organism specific. That is, a library constructed from any tissue from within a single organism should contain the same DNA fragments as those derived from any other tissue. However, libraries generated from different organisms, e.g. those derived from mouse and rat, will be different. Genomic DNA libraries should contain all of the genetic material, whether that material is expressed in a particular tissue type or developmental stage or not. Genomic libraries will therefore contain all DNA sequences: expressed genes, non-expressed genes, exons and introns, promoter and terminator regions and intervening DNA sequences. cDNA libraries are constructed by the conversion of mRNA from a particular tissue sample into DNA fragments that can be cloned into an appropriate vector. cDNA libraries thus contain only the coding sequence of genes expressed in a tissue sample together with small regions of the 5  and 3  untranslated portions of the gene. Consequently, cDNA libraries isolated from different tissues of the same organism may be radically different in their composition. The genes expressed in one tissue type or developmental stage may well be different from those expressed in another tissue type or developmental stage. Additionally, the composition of a cDNA library reflects the relative abundance of mRNA in the original tissue sample. Highly expressed genes will be represented in the library multiple times, whereas genes expressed at a low level will be represented in the library less frequently. 5.1 Genomic Libraries The smallest unit of DNA within a genome is the chromosome. Even in the simplest organisms, however, chromosomes contain an enormous quantity of DNA. For example, the E. coli chromosome contains some 4.6 Mbp (4 600 000 bp) of DNA (Table 5.1). This amount of DNA is far too large to be cloned into any of the vectors currently available (Chapter 3). Therefore it is necessary, and indeed desirable, to fragment the DNA before it is cloned into an appropriate vector. A ‘divide and conquer’ strategy comes into play here, whereby relatively small fragments of the genome can be assigned a specific function whereas the whole genome is somewhat impenetrable. The method of fragmentation plays an important role in the quality of the final library. Ideally, the genomic DNA should be broken up into random and overlapping fragments prior to cloning. Such cleavage would ensure that the library contains representative copies of all DNA fragments present within the genome, and that fragment 186 CLONING A GENE 5 bias is not encountered by the cleavage of DNA at specific sites only. There are two basic mechanisms for cleaving DNA that are used in the construction of genomic libraries. (a) Mechanical shearing. Purified genomic DNA is either passed several times through an narrow-gauge syringe needle or subjected to sonication to break up the DNA into suitable size fragments that can be cloned. Typically, an average DNA fragment size of about 20 kbp is desirable for cloning into λ based vectors. Mechanical methods such as these have the advantage that DNA fragmentation is random, but suffer from the fact that large quantities of DNA are required, and that the average DNA fragmentation size may be quite variable. (b) Restriction enzyme digestion. Restriction enzymes, such as EcoRI, often recognize 6 bp DNA sequences and cleave the DNA within the recognition sequence. On average, a 6 bp DNA sequence will occur approximately every 4000 bp within DNA. Complete digestion of genomic DNA with EcoRI will generate DNA fragments that are generally too small to be useful in genomic library construction. Other restriction enzymes, e.g. NotI, recognize and cleave 8 bp recognition sequences. Such sequences will occur much less commonly within DNA (approximately once every 65 kbp). However, restriction enzyme cleavage to produce DNA fragments suffers as a consequence of the recognition sites themselves. If, by chance, a gene that we would like to clone contains multiple recognition sites for a particular restriction enzyme, then the fragments generated after enzyme digestion may be too small to clone, and consequently the gene may not be represented within a library. To overcome this problem genomic DNA libraries are usually constructed by digesting the genomic DNA with restriction enzymes in such a way that the digestion does not go to completion (Figure 5.1). Partial restriction digests will ensure that not all DNA recognition sequences are cut and, consequently, that the library produced should contain copies of genes that may possess multiple restriction enzyme recognition sequences. In practice, restriction digestion is normally performed using a restriction enzyme, or often two, that recognize and cleave very commonly occurring sequences. For example, as shown in Figure 5.2, high-molecular-weight genomic DNA is partially cleaved with a mixture of the restriction enzymes HaeIII and AluI. Each of these restriction enzymes recognizes a 4 bp DNA sequence. Their recognition sequences should therefore occur, on average, approximately every 256 bp within genomic DNA. The partial digestion, however, limits the number of restriction enzymes sites that are actually cut and leads to 5.1 GENOMIC LIBRARIES 187 Complete digest Partial digest RE RE RE RE RE RE RE RE (b) (a) Figure 5.1. The complete and partial digestion of a DNA fragment using a restriction enzyme. (a) Complete digestion ensures that all restriction enzyme recognition sites (RE) are cut. (b) Partial digestion results in the cleavage of a random subset of the recognition sites. Partial digestion will generate a variety of products as indicated the formation of genomic DNA fragments of a suitable size for cloning. DNA fragments produced in this manner have blunt ends since both HaeIII and AluI cut DNA in a blunt-ended fashion: 5'-GG CC-3'HaeIII: 3'-CC GG-5' 5'-AG CT-3'AluI: 3'-TC GA-5' The blunt ended DNA fragments can prove problematical when attempts are made to clone them. As we have already seen (Chapter 2), the ligation of sticky-ended DNA is considerably more efficient than that in which the DNA ends are blunt. Consequently, it is desirable to generate genomic fragments that contain sticky ends in the cloning process. This can be achieved in one of two ways. • Linkers or adaptors. As shown in Figure 5.2, the blunt ended DNA frag- ments can be ligated to a series of oligonucleotides that either contain the recognition sequence for a restriction enzyme (linkers) or possess one blunt end for ligation to the genomic DNA and an overhanging sticky end for cloning into particular restriction sites (adaptors). In the case shown here, [...]... produce the first cDNA strand (Murakawa et al., 1988) The production of a single strand of cDNA is mRNA 5' – AAAAA–3' Primer 5' – 5' – mRNA TTTTT–3' AAAAA–3' 3'–TTTTT 5' Reverse transcriptase + dNTPs 5' – 3'– AAAAA–3' TTTTT 5' cDNA RNase H 3'– TTTTT cDNA 5' Terminal transferase + dCTP 3'–CCCCC TTTTT 5' cDNA Primer 5' – 5' – GGGGG–3' 3'–CCCCC cDNA GGGGG–3' TTTTT 5' Second strand synthesis 5' – 3'– GGGGG CCCCC... added and uses the oligo-dT as a primer to synthesize a single strand of cDNA in the presence of 194 CLONING A GENE 5 mRNA AAAAA– 3' 5' – Oligo-dT 5' – AAAAA– 3' 3'–TTTTT– 5' Reverse transcriptase + dNTPs mRNA 5' – 3'– AAAAA– 3' TTTTT– 5' cDNA Terminal transferase + dCTP mRNA AAAAACCC– 3' TTTTT– 5' 5' – 3'–CCC cDNA Alkaline sucrose gradient 3'–CCC TTTTT– 5' cDNA Oligo-dG 5' –GGG– 3' 3'–CCC cDNA TTTTT– 5' Reverse... that encode a XhoI 5. 3 DIRECTIONAL cDNA CLONING 197 mRNA 5' – AAAAA– 3' Primer - 5' –GGGCTCGAGTTTTT– 3' 5' – mRNA 5' – 3'– AAAAA- 3' 3'–TTTTTGAGCTCGGG– 5' Reverse transcriptase + dNTPs AAAAA– 3' TTTTTGAGCTCGGG– 5' cDNA mRNA 5' – 3'–CCCCC Terminal transferase + dCTP AAAAACCC– 3' TTTTTGAGCTCGGG– 5' cDNA Alkaline sucrose gradient 3'–CCCCC TTTTTGAGCTCGGG– 5' cDNA Primer - 5' –GGGGAATTCGGGGG– 3' 5' –GGGGAATTCGGGGG–3'... in the target library (genes 3 5) Shown here are agarose gels of the libraries stained with ethidium bromide (EtBr), and then blotted and probed with the DNA sequences for five separate genes The intensity of the band in the probed gels indicates the abundance of the gene in the library Images courtesy of Gerard Brady and Abdulla Bashein, Epistem Ltd analysed the mRNA content of single hemopoietic precursor... using 196 CLONING A GENE 5 mRNA AAAAA–3' 5' – cDNA synthesis 5' – 3'– AAAAA–3' TTTTT 5' Double-stranded cDNA Cloning Promoter TTTTT AAAAA Promoter or AAAAA TTTTT Figure 5. 5 cDNA that is to be expressed must be cloned in a defined orientation so that the promoter element to which it is attached will initiate the transcription of the sense strand of the DNA, rather than the antisense strand an oligo-dG primer... (Figure 5. 5) 5. 3 Directional cDNA Cloning The synthesis of cDNA using modified oligonucleotides to initiate each strand of DNA synthesis allows the insertion of unique restriction enzyme recognition sites at either end of the cDNA so that cloning of the cDNA fragments can only occur in one direction (Figure 5. 6) In the example shown here, the oligo-dT primer also contains additional sequences at the 5 -end... fraction of the total RNA contained within a cell (Table 5. 2) Most eukaryotic protein coding genes are transcribed by RNA polymerase II and the resulting mRNA is usually subjected to a number of post-transcriptional modifications, including the additions of a 7-methylguanosine cap at the 5 -end, and the addition of 100–200 adenine residues (a poly(A) tail) at the 3 -end 192 CLONING A GENE 5 Table 5. 2 The... by using random primers to initiate the first strand of cDNA synthesis The random primers are usually six to nine nucleotides in length and are synthesized to be a mixture of all possible bases at each position (5 -NNNNNN-3 ) Random primers will hybridize at random positions along the mRNA and will serve as starting points for DNA synthesis cDNA cloned by this method, following the synthesis of the second... Table 5. 2 The distribution of RNA molecules within cells In eukaryotes, RNA polymerase II is responsible for the production of approximately 60 per cent of newly synthesized transcripts Due to its instability, however, mRNA accumulates at a level of 10 per cent or less (Brandhorst and McConkey, 1974) RNA type mRNA tRNA rRNA Relative abundance (%) E coli Mammalian 5 15 80 10 15 75 of the transcript (see... enrichment of sequences present only in the tester cDNA library For example, Brady et al st Te riv D er CLONING A GENE 5 er 204 EtBr Gene 2 Gene 3 Subtraction Gene 1 Gene 4 Gene 5 Figure 5. 10 Enrichment of DNA molecules during subtractive hybridization The subtraction of the driver library from the target library results in the elimination of common sequences (genes 1 and 2) and the enrichment of sequences . REACTION 4 3′ 5 3′ 5 3′ 5 3′ 5 3′ 5 Primer 1 5 3′ QR Primer 2 Probe 5 3′ 3′ 5 3′ 5 5 5 Q R 3′ 3′ 5 3′ 5 5 5 Q R 3′ 3′ 5 3′ 5 5 5 Q R Displacement Cleavage Completed synthesis Primer. 178 POLYMERASE CHAIN REACTION 4 AAA-3′ 5 5 3′ mRNA Primer 1 AAA-3′ 5 mRNA 5 3′ DNA 5 3′ 5 3′ 5 3′ 5 5 Primer 1 3′ 3′ Primer 2 3′ 5 5 3′ 3′ 5 Primer 2 RT PCR cycle 1 PCR cycle 2 RT Ta. process of producing a double-stranded cDNA copy of an mRNA molecule is shown in Figure 5. 4. The presence of a polyA tail is unique to mRNA, and provides a mechanism of distinguishing and isolating