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Strachan T, Read AP. Human Molecular Genetics. 2nd edition. New York: Wiley-Liss; 1999.

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Human Molecular Genetics. 2nd edition.

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Chapter 6PCR, DNA sequencing and in vitro mutagenesis

6.1. Basic features of PCR

The polymerase chain reaction (PCR) has revolutionized molecular genetics by permitting rapid cloning and analysis of DNA. Since the first reports describing this new technology in the mid 1980s, there have been numerous applications in both basic and clinical research. Two other fundamental technologies are DNA sequencing and in vitro mutagenesis, both of which can be accomplished using PCR-based and non PCR-based methods.

6.1.1. PCR is a cell-free method of DNA cloning

The standard PCR reaction: selective DNA amplification

PCR is a rapid and versatile in vitro method for amplifying defined target DNA sequences present within a source of DNA. Usually, the method is designed to permit selective amplification of a specific target DNA sequence(s) within a heterogeneous collection of DNA sequences (e.g. total genomic DNA or a complex cDNA population). To permit such selective amplification, some prior DNA sequence information from the target sequences is required. This information is used to design two oligonucleotide primers (amplimers) which are specific for the target sequence and which are often about 15–25 nucleotides long. After the primers are added to denatured template DNA, they bind specifically to complementary DNA sequences at the target site. In the presence of a suitably heat-stable DNA polymerase and DNA precursors (the four deoxynucleoside triphosphates, dATP, dCTP, dGTP and dTTP), they initiate the synthesis of new DNA strands which are complementary to the individual DNA strands of the target DNA segment, and which will overlap each other (Figure 6.1).

Figure 6.1. PCR is an in vitro method for amplifying DNA sequences using defined oligonucleotide primers.

Figure 6.1

PCR is an in vitro method for amplifying DNA sequences using defined oligonucleotide primers. Oligonucleotide primers A and B are complementary to DNA sequences located on opposite DNA strands and flanking the region to be amplified. Annealed primers (more...)

The PCR is a chain reaction because newly synthesized DNA strands will act as templates for further DNA synthesis in subsequent cycles. After about 25 cycles of DNA synthesis, the products of the PCR will include, in addition to the starting DNA, about 105 copies of the specific target sequence, an amount which is easily visualized as a discrete band of a specific size when submitted to agarose gel electrophoresis. A heat-stable DNA polymerase is used because the reaction involves sequential cycles composed of three steps:

  1. Denaturation, typically at about 93–95°C for human genomic DNA.
  2. Reannealing at temperatures usually from about 50°C to 70°C depending on the Tm (see Section 5.2.1) of the expected duplex (the annealing temperature is typically about 5°C below the calculated Tm).
  3. DNA synthesis, typically at about 70–75°C.

Suitably heat-stable DNA polymerases have been obtained from microorganisms whose natural habitat is hot springs. For example, the widely used Taq DNA polymerase is obtained from Thermus aquaticus and is thermostable up to 94°C, with an optimum working temperature of 80°C.

Specificity of amplification and primer design

The specificity of amplification depends on the extent to which the primers can recognize and bind to sequences other than the intended target DNA sequences. For complex DNA sources, such as total genomic DNA from a mammalian cell, it is often sufficient to design two primers about 20 nucleotides long. This is because the chance of an accidental perfect match elsewhere in the genome for either one of the primers is extremely low, and for both sequences to occur by chance in close proximity in the specified direction is normally exceedingly low. Although conditions are usually chosen to ensure that only strongly matched primer-target duplexes are stable, spurious amplification products can nevertheless be observed. This can happen if one or both chosen primer sequences contain part of a repetitive DNA sequence, and primers are usually designed to avoid matching to known repetitive DNA sequences, including large runs of a single nucleotide (Figure 6.2).

Figure 6.2. PCR primer design.

Figure 6.2

PCR primer design.

Accidental matching at the 3′ end of the primer is critically important: spurious products may derive from substantially mismatched primer-target duplexes unless the 3′ end of the primer shows perfect matching. Several strategies can be adopted to optimize reaction specificity:

  • Nested primers. The products of an initial amplification reaction are diluted and used as the target DNA source for a second reaction in which a different set of primers is used, corresponding to sequences located close, but internal, to those used in the first reaction.
  • Hot-start PCR. Mixing of all PCR reagents prior to an initial heat denaturation step allows more opportunity for nonspecific binding of primer sequences. To reduce this possibility, one or more components of the PCR are physically separated until the first denaturation step. A popular approach is to use a specially formulated wax bead designed to fit snugly within a PCR reaction tube. The reaction components minus the enzyme and reaction buffer are added to the tube followed by the molten wax bead which floats on top and then solidifies on cooling. The thermostable polymerase is then added with buffer. At the initial denaturation step the wax melts again and rises to the surface causing all the reaction components to come into contact with each other.
  • Touch-down PCR. Most thermal cyclers can be programed to perform runs in which the annealing temperature is lowered incrementally during the PCR cycling from an initial value above the expected Tm to a value below the Tm. By keeping the stringency of hybridization initially very high, the formation of spurious products is discouraged, allowing the expected sequence to predominate.

DNA labeling by PCR

The standard PCR reaction can be modified to permit incorporation of labeled nucleotides. Two methods are commonly used:

  • Standard PCR-based DNA labeling. The PCR reaction is modified to include one or more labeled nucleotide precursors which become incorporated into the PCR product throughout its length.
  • Primer-mediated 5end labeling. PCR is conducted using a primer in which a labeled group is attached to the 5′ end. As PCR proceeds the primer with its 5′ end-label is incorporated into the PCR product. This method is often used with fluorophore labels during DNA sequencing (see legend to Figure 6.18) and is an example of a general PCR mutagenesis method known as 5add-on mutagenesis which has many applications (see Section 6.4.2 and Figure 6.20A).
Figure 6.18. Cycle sequencing involves linear amplification using a single primer to initiate DNA synthesis.

Figure 6.18

Cycle sequencing involves linear amplification using a single primer to initiate DNA synthesis. Cycle sequencing using the dideoxynucleotide method involves setting up four parallel DNA sequencing reactions in which DNA synthesis occurs, using a mix of (more...)

Figure 6.20. PCR mutagenesis.

Figure 6.20

PCR mutagenesis. (A) 5′ add-on mutagenesis. Primers can be modified at the 5′ end to introduce, for example, a labeled group (Figure 10.24), a sequence containing a suitable restriction site (Figure 20.12) or a phage promoter to drive (more...)

6.1.2. The major advantages of PCR as a cloning method are its rapidity, sensitivity and robustness

Because of its simplicity, PCR is a popular technique with a wide range of applications which depend on essentially three major advantages of the method.

Speed and ease of use

DNA cloning by PCR can be performed in a few hours, using relatively unsophisticated equipment. Typically, a PCR reaction consists of 30 cycles containing a denaturation, synthesis and reannealing step, with an individual cycle typically taking 3–5 min in an automated thermal cycler. This compares favorably with the time required for cell-based DNA cloning, which may take weeks. Clearly, some time is also required for designing and synthesizing oligonucleotide primers, but this has been simplified by the availability of computer software for primer design and rapid commercial synthesis of custom oligonucleotides. Once the conditions for a reaction have been tested, the reaction can then be repeated simply.

Sensitivity

PCR is capable of amplifying sequences from minute amounts of target DNA, even the DNA from a single cell (Li et al., 1988). Such exquisite sensitivity has afforded new methods of studying molecular pathogenesis and has found numerous applications in forensic science, in diagnosis, in genetic linkage analysis using single-sperm typing and in molecular paleontology studies, where samples may contain minute numbers of cells. However, the extreme sensitivity of the method means that great care has to be taken to avoid contamination of the sample under investigation by external DNA, such as from minute amounts of cells from the operator.

Robustness

PCR can permit amplification of specific sequences from material in which the DNA is badly degraded or embedded in a medium from which conventional DNA isolation is problematic. As a result, it is again very suitable for molecular anthropology and paleontology studies, for example the analysis of DNA recovered from archaeological remains. It has also been used successfully to amplify DNA from formalin-fixed tissue samples, which has important applications in molecular pathology and, in some cases, genetic linkage studies.

6.1.3. The major disadvantages of PCR are the general requirement for prior target sequence information, short size and limiting amounts of product, and infidelity of DNA replication

Despite its huge popularity, PCR has certain limitations as a method for selectively cloning specific DNA sequences.

Need for target DNA sequence information

In order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is necessary. This normally means that the DNA region of interest has been partly characterized previously, often following cell-based DNA cloning. However, a variety of techniques have been developed that reduce or even exclude the need for prior DNA sequence information concerning the target DNA, when certain aims are to be met. For example, previously uncharacterized DNA sequences can sometimes be cloned using PCR with degenerate oligonucleotides if they are members of a gene or repetitive DNA family at least one of whose members has previously been characterized. In some cases, PCR can be used effectively without any prior sequence information concerning the target DNA to permit indiscriminate amplification of DNA sequences from a source of DNA that is present in extemely limited quantities (Section 6.2.4). Therefore, although PCR can be applied to ensure whole genome amplification, it does not have the advantage of cell-based DNA cloning in offering a way of separating the individual DNA clones comprising a genomic DNA library.

Short size and limiting amounts of PCR product

A clear disadvantage of PCR as a DNA cloning method has been the size range of the DNA sequences that can be cloned. Unlike cell-based DNA cloning where the size of cloned DNA sequences can approach 2 Mb (Section 4.3.4), reported DNA sequences cloned by PCR have typically been in the 0.1–5 kb size range, often at the lower end of this scale. Although small segments of DNA can usually be amplified easily by PCR, it becomes increasingly more difficult to obtain efficient amplification as the desired product length increases. Recently, however, conditions have been identified for effective amplification of longer targets, including a 42-kb product from the bacteriophage λ genome. Often, the conditions for long range PCR involve a combination of modifications to standard conditions with a two-polymerase system. This provides optimal levels of DNA polymerase and 3′ → 5′ exonuclease activity which serves as a proofreading mechanism (see Box 6.1).

Box Icon

Box 6.1

Proofreading by DNA polymerase-associated 3′ → 5′ exonuclease activity. The fidelity of DNA replication in vivo is extremely high: during replication of mammalian genomes, for example, only one base in about 3 × 109 is (more...)

The amount of PCR product obtained in a single reaction is also much more limited than the amount that can be obtained using cell-based cloning where scale-up of the volumes of cell cultures is possible. The efficiency of a PCR reaction will vary from template to template and according to various factors that are required to optimize the reaction but typically only comparatively small amounts of product are achieved.

Infidelity of DNA replication

Cell-based DNA cloning involves DNA replication in vivo, which is associated with a very high fidelity of copying because of proofreading mechanisms (see Box 6.1). However, when DNA is replicated in vitro the copying error rate is considerably greater. Of the heat-stable DNA polymerases required for PCR, the most widely used is Taq DNA polymerase derived from T. aquaticus. This DNA polymerase, however, has no associated 3′ → 5′ exonuclease to confer a proofreading function, and the error rate due to base misincorporation during DNA replication is rather high: for a 1 kb sequence that has undergone 20 effective cycles of duplication, approximately 40% of the new DNA strands synthesized by PCR using this enzyme will contain an incorrect nucleotide resulting from a copying error. This means that, even if the PCR reaction involves amplification of a single DNA sequence, the final product will be a mixture of extremely similar, but not identical DNA sequences.

Despite the errors due to replication in vitro, DNA sequencing of the total PCR product may give the correct sequence. This is because, although individual DNA strands in the PCR product often contain incorrect bases, the incorporation of incorrect bases is essentially random. As a result, for each base position, the contribution of one incorrect base on one or more strands is overwhelmed by the contributions from the huge majority of strands which will have the correct sequence. What it does mean, however, is that further analysis of the product may be difficult. If the PCR product is to be cloned in cells (e.g. to facilitate DNA sequencing or to permit functional studies in a cell-based expression system), transformation selects for a single molecule, and the cell clones chosen to be amplified will contain identical molecules, each the same as a single starting molecule which may well have the incorrect DNA sequence because of a copying error during PCR amplification. As a result, several individual clones may need to be sequenced in order to determine the correct (consensus) sequence, before selecting one with the authentic sequence for subsequent experiments.

More recently, the problem of infidelity of DNA replication during the PCR reaction has been considerably reduced by using alternative heat-stable DNA polymerases which have associated 3′–5′ exonuclease activity. For example, the Pyrococcus furiosus DNA polymerase is becoming more widely used because of the proofreading conferred by its associated 3′–5′ exonuclease activity (Cline et al., 1996). The resulting PCR product has a much lower level of mutations introduced by copying errors: for a 1 kb segment of DNA that has undergone 20 effective cycles of duplication, about 3.5% of the DNA strands in the product carry an altered base.

6.1.4. Cell-based cloning of PCR amplification products is often required to permit subsequent structural and functional studies

The amount of material that can be cloned in a single PCR reaction is limited, and it is time-consuming and expensive to repeat the same PCR reaction many times to achieve large quantities of the desired DNA. In addition, the PCR product may not be in a suitable form that will permit some subsequent studies. As a result, it is often convenient to clone the PCR product in a cell-based cloning system in order to obtain large quantities of the desired DNA and to permit a variety of analyses. As described in the previous section, it is important to verify that the sequence of the cloned product is representative of the original PCR product.

Various plasmid cloning systems are used to propagate PCR-cloned DNA in bacterial cells. Once cloned, the insert can be cut out using suitable restriction nucleases and transferred into other plasmids which may have specialized usages in permitting expression to give an RNA product, or to provide large quantities of a protein, etc. Several thermostable polymerases including Taq DNA polymerase have a terminal deoxynucleotidyl transferase activity which selectively modifies PCR-generated fragments by adding a single nucleotide, generally adenine, to the 3′ ends of amplified DNA fragments. The resulting overhangs can make it difficult to clone PCR products and a variety of approaches are commonly used to facilitate cloning, including the use of vectors with overhanging T residues in their cloning site polylinker and the use of ‘polishing’ enzymes such as T4 polymerase or Pfu polymerase which can remove the overhanging single nucleotides (Figure 6.3).

Figure 6.3. Cloning of PCR products in bacterial cells.

Figure 6.3

Cloning of PCR products in bacterial cells. PCR products frequently have an overhanging adenosine at their 3′ ends (see text). The T-A cloning system has a polylinker system with complementary thymine overhangs to facilitate cloning. An alternative (more...)

6.2. Applications of PCR

Although PCR was first developed only a decade and a half ago, the simplicity and the versatility of the technique have ensured that it is among the most ubiquitous of molecular genetic methodologies, with a wide range of general applications (Figure 6.4).

Figure 6.4. PCR has numerous general applications.

Figure 6.4

PCR has numerous general applications. The figure illustrates general applications. Specific applications are described in separate chapters. Genome walking means accessing uncharacterized DNA starting from a neighboring characterized sequence.

6.2.1. PCR enables rapid amplification of template DNA for screening of uncharacterized mutations

Because of its rapidity and simplicity, PCR is ideally suited to providing numerous DNA templates for mutation screening. Partial DNA sequences, at the genomic or the cDNA level, from a gene associated with disease, or some other interesting phenotype, immediately enable gene-specific PCR reactions to be designed. Amplification of the appropriate gene segment then enables rapid testing for the presence of associated mutations in large numbers of individuals. By contrast, cell-based DNA cloning of the gene from numerous different individuals is far too slow and labor-intensive to be considered as a serious alternative.

Typically, the identification of exon-intron boundaries and sequencing of the ends of introns of a gene of interest offers the possibility of genomic mutation screening. Individual exon-specific amplification reactions are developed by designing primers which recognize intronic sequences located close to the exon-intron boundary (Figure 6.5A). The resulting PCR products are then analyzed by rapid mutation-screening methods, in which the optimal size for mutation screening is usually about 200 bp (see Section 15.5.1). Conveniently, the average size of a human exon is about 180 bp but, in the case of very large exons, it is usual to design a series of primers to generate overlapping exonic products. PCR can also quickly provide amplified cDNA sequences for mutation screening. Such cDNA mutation screening may be the only way in which mutations can be screened if the exon-intron organization of a gene has not been established. To do this, mRNA is isolated from a convenient source of tissue, such as blood cells, converted into cDNA using reverse transcriptase and the cDNA is used as a template for a PCR reaction. This version of the standard genomic PCR reaction is consequently often referred to as RT-PCR (reverse transcriptase-PCR; Figure 6.5B). Clearly, the method is ideally suited to genes expressed at high levels in easily accessible cells, such as blood cells. However, as a result of low level ectopic transcription of genes in all tissues, it has also been applied to transcript analysis of genes which are not significantly expressed in blood cells, such as the dystrophin (DMD) gene (Chelly et al., 1989).

Figure 6.5. PCR products for gene mutation screening are obtained from genomic DNA using intron-specific primers flanking exons or by RT-PCR.

Figure 6.5

PCR products for gene mutation screening are obtained from genomic DNA using intron-specific primers flanking exons or by RT-PCR. (A) Genomic DNA. Exons 1–4 can be amplified separately from genomic DNA using pairs of intron-specific primers 1F (more...)

6.2.2. PCR permits rapid genotyping for polymorphic markers

Restriction site polymorphisms (RSPs) result in alleles possessing or lacking a specific restriction site. Such polymorphisms can be typed using Southern blot hybridization. A DNA probe representing the locus is hybridized against genomic DNA samples that have been digested with the appropriate restriction enzyme and size-fractionated by agarose gel electrophoresis. The resulting RFLPs have two alleles corresponding to the presence or absence of the restriction site (Section 5.3.3). As a convenient alternative to RFLPs, PCR can type RSPs by simply designing primers using sequences which flank the polymorphic restriction site, amplifying from genomic DNA, then cutting the PCR product with the appropriate restriction enzyme and separating the fragments by agarose gel electrophoresis (Figure 6.6).

Figure 6.6. Restriction site polymorphisms can easily be typed by PCR as an alternative to laborious RFLP assays.

Figure 6.6

Restriction site polymorphisms can easily be typed by PCR as an alternative to laborious RFLP assays. Alleles 1 and 2 are distinguished by a polymorphism which alters the nucleotide sequence of a specific restriction site for restriction nuclease R: allele (more...)

Short tandem repeat polymorphisms (STRPs), also called microsatellite markers, consist of a short sequence, typically from one to four nucleotides long, that is tandemly repeated several times, and often characterized by many alleles. For example, (CA) n /(TG) n repeats are often polymorphic when n exceeds 12, and have been widely used as polymorphic markers in the human genome (see below). Increasingly, however, trinucleotide and tetranucleotide marker polymorphisms are being typed. In each case the STRPs can be typed conveniently by PCR. Primers are designed from sequences known to flank a specific STRP locus, permitting PCR amplification of alleles whose sizes differ by integral repeat units (Figure 6.7). The PCR products can then be size-fractionated by polyacrylamide gel electrophoresis. The PCR normally includes a radioactive or fluorescent nucleotide precursor which becomes incorporated into the small PCR products and facilitates their detection. To ensure adequate size fractionation of alleles, the PCR products are denatured prior to electrophoresis. An example of the use of a CA repeat marker is shown in Figure 6.8.

Figure 6.7. PCR can be used to type short tandem repeat polymorphisms (STRPs).

Figure 6.7

PCR can be used to type short tandem repeat polymorphisms (STRPs). The example illustrates typing of a (CA)/(TG) dinucleotide repeat polymorphism which has three alleles as a result of variation in the number of the (CA) repeats. On the autoradiograph (more...)

Figure 6.8. Example of typing for a CA repeat.

Figure 6.8

Example of typing for a CA repeat. The example illustrated shows typing of members of a large family with the (CA)/(TG) marker D17S800. Arrows to the left mark the top (main) band seen in different alleles 1–7. Note that individual alleles show (more...)

6.2.3. A wide variety of PCR-based methods can be used to assay for known mutations

PCR is a very rapid and valuable tool for detecting pathogenic mutations and other mutations of interest. The examples below illustrate some popular methods.

Allelic discrimination by size or susceptibility to restriction enzyme

Small insertions or deletions (such as the three nucleotide deletion in the common cystic fibrosis (CFTR) allele, F508del) can be simply detected by designing primers from regions closely flanking the mutation site and distinguishing the normal and mutant alleles by size on polyacrylamide or agarose gels. If the mutation changes a restriction site, mutant and normal alleles can be distinguished by amplifying across the mutant site and digesting the PCR product with relevant restriction endonuclease, exactly as in Figure 6.6.

Allelic discrimination by susceptibility to an artificially introduced restriction site

Even if the mutation does not result in a restriction site difference, it may be possible to exploit the difference between normal and mutant alleles by amplificationcreated restriction site PCR. This is a form of mismatched primer mutagenesis (see Section 6.4.2) in which a primer is deliberately designed from sequence immediately adjacent to, but not encompassing, the restriction site. The primer is deliberately designed to have a mismatched nucleotide which together with the sequence of the mutant site creates a restriction site not present in normal alleles (see Figure 17.2 for a specific example).

Allele-specific PCR (ARMS test)

Oligonucleotide primers can be designed so as to discriminate between target DNA sequences that differ by a single nucleotide in the region of interest. This is a form of allele-specific PCR, the PCR equivalent of the allele-specific hybridization which is possible with ASO probes (Section 5.3.1). In the case of allele-specific hybridization, alternative ASO probes are designed to have differences in a central segment of the sequence (to maximize thermodynamic instability of mismatched duplexes). However, in the case of allele-specific PCR, ASO primers are designed to differ at the nucleotide that occurs at the extreme 3terminus. This is so because the DNA synthesis step in a PCR reaction is crucially dependent on correct base-pairing at the 3′ end (Figure 6.9). This method can be used to type specific alleles at a polymorphic locus, but has found particular use as a method for detecting a specific pathogenic mutation, the so-called amplification refractory mutation system (ARMS; Newton et al., 1989).

Figure 6.9. Correct base-pairing at the 3′ end of PCR primers is the basis of allele-specific PCR.

Figure 6.9

Correct base-pairing at the 3′ end of PCR primers is the basis of allele-specific PCR. The allele-specific oligonucleotide primers ASP1 and ASP2 are designed to be identical to the sequence of the two alleles over a region preceding the position (more...)

Mutation detection using the 5′ → 3′ exonuclease activity of Taq DNA polymerase (TaqMan™ assay)

Taq polymerase does not possess a proofreading 3′ → 5′ exonuclease activity but does possess a 5′ → 3′ exonuclease activity. This property can be exploited to facilitate detection of specific alleles (Holland et al., 1991; Lee et al., 1993). Such an assay involves hybridization of three primers, the third primer being intended to bind just downstream of one of the conventional primers which should be allele-specific. The additional primer carries a blocking group at the 3′ terminal nucleotide so that it cannot prime new DNA synthesis and at its 5′ end carries a labeled group. In modern versions of the assay, the label is a fluorogenic group and the third primer also carries a quencher group (see Figure 6.10). If the upstream primer which is bound to the same strand is able to prime successfully, Taq DNA polymerase will extend a new DNA strand until it encounters the third primer in which case its 5′ → 3′ exonuclease will degrade the primer causing release of separate nucleotides containing the dye and the quencher, and an observable increase in fluorescence.

Figure 6.10. The TaqMan™ 5′ exonuclease assay.

Figure 6.10

The TaqMan™ 5′ exonuclease assay. In addition to two conventional PCR primers, P1 and P2, which are specific for the target sequence, a third primer, P3, is designed to bind specifically to a site on the target sequence downstream of the (more...)

6.2.4. Degenerate oligonucleotide primers and primers specific for ligated linker sequences permit co-amplification of sequence families, or even indiscriminate amplification

DOP-PCR (degenerate oligonucleotide-primed PCR) is a form of PCR which is deliberately designed to permit possible amplification of several products. The two primers may be partially degenerate oligonucleotides, composed of panels of oligonucleotide sequences that have the same base at certain nucleotide positions, but are different at others. As a result, there may be comparatively many primer binding sites in the source DNA. This provides a means of searching for a new or uncharacterized DNA sequence that belongs to a family of related sequences either within or between species. Note: the use of such primers also provides a way of cloning a gene when only a limited portion of amino acid sequence is known for the product (Figure 6.11).

Figure 6.11. DOP-PCR can permit cDNA cloning using degenerate oligonucleotides.

Figure 6.11

DOP-PCR can permit cDNA cloning using degenerate oligonucleotides. The figure illustrates cloning of a cDNA for porcine urate oxidase using degenerate oligonucleotides corresponding to a known amino acid sequence. The sense primer was constructed to correspond (more...)

DOP-PCR can also be used to permit comparatively indiscriminate amplification of target DNA. Primer sequences with random sequences can bind to numerous locations in the template DNA and permit a form of whole-genome amplification (Zhang et al., 1992; Cheung and Nelson, 1996). This can be advantageous where the amount of starting DNA may be limiting (as in the case of extracts from ancient DNA samples, microdissected chromosome bands, single cell typing, etc.), and PCR amplification of essentially all sequences increases the amount of DNA for study.

Linker-primed PCR (ligation adaptor PCR)

Another way of enabling amplification of essentially all DNA sequences in a complex DNA mixture involves first ligating a known sequence to all fragments. To do this, the target DNA population is digested with a suitable restriction endonuclease, and double-stranded oligonucleotide linkers (also called adaptors) with a suitable overhanging end are ligated to the ends of target DNA fragments. Amplification is then performed using oligonucleotide primers which are specific for the linker sequences. In this way, all fragments of the DNA source which are flanked by linker oligonucleotides can be amplified (Figure 6.12).

Figure 6.12. Linker-primed PCR permits indiscriminate amplification of DNA sequences in a complex target DNA.

Figure 6.12

Linker-primed PCR permits indiscriminate amplification of DNA sequences in a complex target DNA. The linker (adaptor) molecule is a double-stranded oligonucleotide formed by ligating two single-stranded oligonucleotides which are complementary in sequence (more...)

6.2.5. Anchored PCR uses a target-specific primer and a universal primer for amplifying sequences adjacent to a known sequence

It is often desirable to be able to amplify previously uncharacterized DNA sequences that neighbor a known DNA sequence, either at the genomic or cDNA level. To do this a form of anchored PCR is used (see Figure 6.13). One of the primers is specific for the target sequence and the second primer is specific for a common sequence that can be introduced in different ways, such as by using a linker-primer method as described in the previous section, or by using primers that are modified at the 5′ end so as to introduce a novel sequence.

Figure 6.13. ‘Genome walking’ by anchored PCR.

Figure 6.13

‘Genome walking’ by anchored PCR. The target may be a complex source of DNA comprised of many fragments to which an anchor sequence is attached, for example a double-stranded oligonucleotide linker. The idea is to use a primer specific (more...)

6.3. DNA sequencing

6.3.1. DNA sequencing usually involves enzymatic DNA synthesis in the presence of base-specific dideoxynucleotide chain terminators

Formerly, chemical DNA sequencing methods were often employed, using base-specific chemical modification and subsequent cleavage of the DNA. Currently, however, the vast majority of DNA sequencing is carried out using an enzymatic method: the DNA to be sequenced is provided in a single-stranded form from which DNA polymerase synthesizes new complementary DNA strands. Usually, the single-stranded DNA template is obtained using a cloning system which permits recovery of single-stranded recombinant DNA, as with M13 or phagemid cloning systems (Section 4.4.1 and Figure 4.17). The subsequent DNA sequencing reactions involve DNA synthesis using one or more labeled nucleotides and a universal sequencing primer that is complementary to the vector sequence flanking the cloning site (Figure 6.14).

Figure 6.14. A universal sequencing primer can be used to sequence many different template DNAs.

Figure 6.14

A universal sequencing primer can be used to sequence many different template DNAs. DNA templates for DNA sequencing are often single-stranded recombinant DNA molecules. Different clones will often contain different inserts within the same vector molecule. (more...)

In addition to the normal nucleotide precursors, DNA synthesis is carried out in the presence of base-specific dideoxynucleotides (ddNTPs). The latter are analogs of the normal dNTPs but differ in that they lack a hydroxyl group at the 3′ carbon position as well as the 2′ carbon (Figure 6.15). A dideoxynucleotide can be incorporated into the growing DNA chain by forming a phosphodiester bond between its 5′ carbon atom and the 3′ carbon of the previously incorporated nucleotide. However, since ddNTPs lack a 3′ hydroxyl group, any ddNTP that is incorporated into a growing DNA chain cannot participate in phosphodiester bonding at its 3′ carbon atom, thereby causing abrupt termination of chain synthesis.

Figure 6.15. Structure of a dideoxynucleotide, 2′, 3′ dideoxy CTP.

Figure 6.15

Structure of a dideoxynucleotide, 2′, 3′ dideoxy CTP. Note that the hydroxyl group which is attached to carbon 3′ in normal nucleotides (see Figure 1.2) is replaced by a hydrogen atom.

Four parallel base-specific reactions are conducted using a mix of all four dNTPs and also a small proportion of one of the four ddNTPs. By setting the concentration of the ddNTP to be very much lower than that of its normal dNTP analog, chain termination will occur randomly at one of the many positions containing the base in question. Each reaction is therefore a partial reaction: chain termination occurs randomly at one of the possible bases in any one DNA strand. However, the DNA to be sequenced in a DNA sequencing reaction is a population of (usually) identical molecules. As a result, each one of the four base-specific reactions will generate a collection of labeled DNA fragments of different sizes, with a common 5end but variable 3ends (the common 5′ end is defined by the sequencing primer and the 3′ ends which terminate with the chosen ddNTP are variable because the insertion of the dideoxynucleotide occurs randomly at one of the many different positions that will accept that specific base - Figure 6.16A).

Figure 6.16. Dideoxy DNA sequencing relies on synthesizing new DNA strands from a single-stranded DNA template and random incorporation of a base-specific dideoxynucleotide to terminate chain synthesis.

Figure 6.16

Dideoxy DNA sequencing relies on synthesizing new DNA strands from a single-stranded DNA template and random incorporation of a base-specific dideoxynucleotide to terminate chain synthesis. (A) Principle of dideoxy sequencing. The sequencing primer binds (more...)

Fragments that differ in size by even a single nucleotide can be separated on a denaturing polyacrylamide gel. The differently sized fragments can be detected by incorporating labeled groups into the reaction products, either by incorporating labeled nucleotides or by using a primer with a labeled group. The sequence can then be read off by reading from the bottom of the gel to the top, a direction that gives the 5′ → 3′ sequence of the complementary strand of the provided DNA template (see Figure 6.16B).

6.3.2. DNA sequencing is increasingly being conducted using fluorescent labeling systems and automated detection systems

Traditional dideoxy sequencing methods have employed radioisotope labeling: the dNTP mix contains a proportion of radiolabeled nucleotides which are incorporated within the growing DNA chains. Following electrophoresis, the gel is dried and an autoradiographic film is placed in contact with the dried gel. After a suitable exposure time, the film is developed, giving a characteristic pattern of dark bands (Figure 6.16B). 32P-labeled nucleotides are not very suitable for this purpose: the high energy β-radiation causes considerable scattering of the signal, leading to diffuse bands. Instead, 35S- or 33P-labeled nucleotides have been used.

Large-scale DNA sequencing efforts are dependent on improving efficiency by partial automation of the technologies involved. One major improvement in recent years has been the development of automated procedures for fluorescent DNA sequencing (Wilson et al., 1990). These procedures generally use primers or dideoxynucleotides to which are attached fluorophores (chemical groups capable of fluorescing - see Section 5.1.2). During electrophoresis, a monitor detects and records the fluorescence signal as the DNA passes through a fixed point in the gel (Figure 6.17A). The use of different fluorophores in the four base-specific reactions means that, unlike conventional DNA sequencing, all four reactions can be loaded into a single lane. The output is in the form of intensity profiles for each of the differently colored fluorophores (Figure 6.17B), but the information is simultaneously stored electronically. This precludes transcription errors when an interpreted sequence is typed by hand into a computer file. Recent advances in technology mean that the accuracy of DNA sequencing using automated methods is acceptably high.

Figure 6.17. Automated DNA sequencing using fluorescent primers.

Figure 6.17

Automated DNA sequencing using fluorescent primers. (A) Principles of automated DNA sequencing. Automated DNA sequencing involves loading all four reaction products into single lanes of the electrophoresis gel and capture of sequence data during the electrophoresis (more...)

6.3.3. PCR-amplified products are often used for DNA sequencing

Cycle sequencing

Double-stranded DNA templates can be used in standard dideoxy sequencing by denaturing the DNA prior to binding the oligonucleotide primer. However, the quality of sequences from initially double-stranded DNA templates is often poor. Cycle sequencing, also called linear amplification sequencing, is a kind of PCR sequencing approach which overcomes this problem. Like the standard PCR reaction, it uses a thermostable DNA polymerase and a temperature cycling format of denaturation, annealing and DNA synthesis. The difference is that cycle sequencing employs only one primer and includes a ddNTP chain terminator in the reaction. The use of only a single primer means that unlike the exponential increase in product during standard PCR reactions, the product accumulates linearly (see Figure 6.18). Because the product accumulates during the reaction, and because of the high temperature at which the sequencing reactions are carried out, and the multiple heat denaturation steps, small amounts of double-stranded plasmids, cosmids, λDNA and PCR products may be sequenced reliably without a separate heat denaturation step.

6.3.4. DNA microarray technology permits an alternative approach to DNA sequencing

DNA sequencing can be accomplished by hybridization of the target DNA to a series of oligonucleotides of known sequence, usually about 7–8 nucleotides long. If the hybridization conditions are specific, it is possible to check which oligonucleotides are positive by hybridization, feed the results into a computer and use a program to look for sequence overlaps in order to establish the required DNA sequence. DNA microarrays have permitted sequencing by hybridization to oligonucleotides on a large scale (Southern, 1996) and in a test system, the sequence of human mtDNA previously first determined in 1981 was recently re-sequenced by DNA microarray hybridization. This type of technology is increasing in importance for assessing sequence variation over at least modest lengths of DNA and diagnostic applications in mutation analysis are proliferating (Hacia, 1999; Section 17.1.4).

6.4. In vitro site-specific mutagenesis

Mutagenesis is a fundamentally important DNA technology which seeks to change the base sequence of DNA and test its effect on gene or DNA function. The mutagenesis can be conducted in vivo (in studies of model organisms, or cultured cells) or in vitro and the mutagenesis can be directed to a specific site in a pre-determined way (site-directed mutagenesis), or can be random. In the case of in vivo mutagenesis, for example, gene targeting offers exquisite site-directed mutagenesis within living cells (Section 21.3.1) while exposure of male mice to high levels of a powerful mutagen such as ethyl nitrosurea (ENU) and subsequent mating of the mice offers a form of random mutagenesis which can be important in generating new mutants (Section 21.4.1).

In vitro mutagenesis can involve essentially random approaches to mutagenesis, which may be valuable in producing libraries of new mutants. In addition, if a gene has been cloned and a functional assay of the product is available, it is also very useful to be able to employ a form of in vitro mutagenesis which results in alteration of a specific amino acid or small component of the gene product in a predetermined way.

6.4.1. Oligonucleotide mismatch mutagenesis is a popular method of introducing a predetermined single nucleotide change into a cloned gene

Many in vitro assays of gene function wish to gain information on the importance of individual amino acids in the encoded polypeptide. This may be relevant when attempting to assess whether a particular missense mutation found in a known disease gene is pathogenic, or just generally in trying to evaluate the contribution of a specific amino acid to the biological function of a protein. A popular general approach involves cloning the gene or cDNA into an M13 or phagemid vector which permits recovery of single-stranded recombinant DNA (Section 4.4.1). A mutagenic oligonucleotide primer is then designed whose sequence is perfectly complementary to the gene sequence in the region to be mutated, but with a single difference: at the intended mutation site it bears a base that is complementary to the desired mutant nucleotide rather than the original. The mutagenic oligonucleotide is then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. The newly formed heteroduplex is used to transform cells, and the desired mutant genes can be identified by screening for the mutation (see Figure 6.19).

Figure 6.19. Oligonucleotide mismatch mutagenesis can create a desired point mutation at a unique predetermined site within a cloned DNA molecule.

Figure 6.19

Oligonucleotide mismatch mutagenesis can create a desired point mutation at a unique predetermined site within a cloned DNA molecule. The figure illustrates only one of many different methods of cell-based oligonucleotide mismatch mutagenesis (for alternative PCR-based (more...)

Other small-scale mutations can also be introduced in addition to single nucleotide substitutions. For example, it is possible to introduce a three-nucleotide deletion that will result in removal of a single amino acid from the encoded polypeptide, or an insertion that adds a new amino acid. Provided the mutagenic oligonucleotide is long enough, it will be able to bind specifically to the gene template even if there is a considerable central mismatch. Still larger mutations can be introduced by using cassette mutagenesis in which case a specific region of the original sequence of the original gene is deleted and replaced by oligonucleotide cassettes (Bedwell et al., 1989).

6.4.2. PCR can be used to couple desired sequences or chemical groups to a target sequence and to produce specific pre-determined mutations in DNA sequences

In addition to long-established nonPCR based methods, site-directed mutagenesis by PCR has become increasingly popular and various strategies have been devised to enable base substitutions, deletions and insertions (see below and Newton and Graham, 1997). In addition to producing specific predetermined mutations in a target DNA, a form of mutagenesis known as 5′ add-on mutagenesis permits addition of a desired sequence or chemical group in much the same way as can be achieved using ligation of oligonucleotide linkers (see Box 4.2).

5′ Add-on mutagenesis

This is a commonly used practice in which a new sequence or chemical group is added to the 5′ end of a PCR product by designing primers which have the desired specific sequence for the 3′ part of the primer while the 5′ part of the primer contains the novel sequence or a sequence with an attached chemical group. The extra 5′ sequence does not participate in the first annealing step of the PCR reaction (only the 3′ part of the primer is specific for the target sequence), but it subsequently becomes incorporated into the amplified product, thereby generating a recombinant product (Figure 6.20A). Various popular alternatives for the extra 5′ sequence include: (i) a suitable restriction site which may facilitate subsequent cell-based DNA cloning; (ii) a functional component, e.g. a promoter sequence for driving expression (see Figure 17.9 for an example); a modified nucleotide containing a reporter group or labeled group, such as a biotinylated nucleotide (see Figure 10.24 for an example) or fluorophore.

Mismatched primer mutagenesis

The primer is designed to be only partially complementary to the target site but in such a way that it will still bind specifically to the target. Inevitably this means that the mutation is introduced close to the extreme end of the PCR product. As described in Section 6.2.3 this approach may be exploited to introduce an artificial diagnostic restriction site that permits screening for a known mutation. Mutations can also be introduced at any point within a chosen sequence using mismatched primers. Two mutagenic reactions are designed in which the two separate PCR products have partially overlapping sequences containing the mutation. The denatured products are combined to generate a larger product with the mutation in a more central location (Higuchi, 1990; Figure 6.20B).

Further reading

  1. Ehrlich HA (1989) PCR Technology. Principles and Applications for DNA Amplification. Stockton Press, New York.
  2. Ehrlich H A, Gelfand D, Sninsky J J. Recent advances in the polymerase chain reaction. Science. (1991);252:1643–1651. [PubMed: 2047872]
  3. Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA.
  4. Ling M M, Robinson B H. Approaches to DNA mutagenesis: an overview. Analyt. Biochem. (1997);254:157–178. [PubMed: 9417773]
  5. McPherson MJ, Taylor GR, Quirke P (1991) PCR: a Practical Approach. IRL Press, Oxford.
  6. Newton CR, Graham A (1997) PCR, 2nd edn. BIOS Scientific Publishers, Oxford.

References

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  2. Chelly J, Concordet J P, Kaplan J C, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc. Natl Acad. Sci. USA. (1989);86:2617–2621. [PMC free article: PMC286968] [PubMed: 2495532]
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  6. Hacia J G. Resequencing and mutational analysis using oligonucleotide microarrays. Nat. Genet. (1999);21 (1 Suppl.):42–47. [PubMed: 9915500]
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  14. Newton CR, Graham A (1997) PCR, 2nd edn, pp. 75–84. BIOS Scientific Publishers, Oxford.
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