key: cord-0034650-ufk3hsbi
authors: nan
title: A Brief Comparison Between In Vivo DNA Replication and In Vitro PCR Amplification
date: 2008
journal: Principles and Technical Aspects of PCR Amplification
DOI: 10.1007/978-1-4020-6241-4_2
sha: 60dae494077004ff268f3085bde38efde30dee27
doc_id: 34650
cord_uid: ufk3hsbi

In principle, PCR generates large quantities of DNA from a minute amount of nucleic acid starting material using a methodology similar to (but much simpler than) that seen in living cells. For living cells, in vivo DNA synthesis is dependent upon a well defined but complex set of enzymes and co-factors, which have evolved to act in a concerted fashion during the synthetic phase (S-phase) of the cell cycle. In comparison, PCR facilitates in vitro DNA synthesis in a much simpler fashion, making use of a smaller set of defined ingredients and reaction conditions involving relatively high temperatures. The range of factors contributing to successful PCR amplification is reviewed below.

In vivo, the double stranded DNA molecule is organized into a highly condensed structure with an impressive molecular density. The primary double helix structure is embedded into spherical nucleosomes (11 nm in diameter) via an interaction with chaperone or histone proteins. These nucleosomes are in turn ordered into 30 nm chromatin fibers which are looped into macrostructures called chromatin. This chromatin may be further condensed into heterochromatin, providing the framework that generates the final eukaryotic chromosomal structure. What is shown is a beads-on-a-string model of chromatin with a linker length of 20 nucleotide pairs in three dimensions (From http://en.wikipedia.org/wiki/Chromatin) DNA Isolated patches Add core histones Add histone H1.

Add further scaffold proteins.

Add further scaffold proteins.

Genes under active transcription.

The Nucleosome "Bead-on-a-String"

The 30 nm Fibre Active Chromosome The Metaphase Chromosome is generally considered to be somewhat simpler, with microorganisms lacking a visible nucleus and containing only one (circular) chromosomal copy per cell. However, it is known that prokaryotic DNA is also associated with a variety of histone-like proteins, which intimately interact with the cellular DNA, protecting it and conferring complex structure (though this structure is generally less ordered or regular than that associated with eukaryotic chromosomes). Therefore, one of the major requirements for DNA replication in in vivo systems is the systematic uncoiling of specific regions of genomic DNA in order to expose single strands of DNA ready for replication. In both eukaryotic and prokaryotic kingdoms this process is achieved via specific DNA topoisomerases, helicases and gyrases, which act together to help uncoil the double stranded DNA. In contrast, the uncoiling of DNA during PCR amplification is not enzymatically controlled but is in fact achieved using a far less complicated procedure, i.e. the DNA to be amplified is usually chemically extracted from its host chaperone proteins, and the residual tertiary or secondary structure removed by heating the naked DNA. This heating step also provides the mechanism by which the two DNA strands are separated (melted) into single strands ready for PCR amplification. As a further point, whereas in vivo DNA chromosomal replication involves the replication of many millions of nucleotides, PCR amplification products are generally designed to be shorter than 1,000 bp in length (a parameter largely imposed on the PCR amplification process by the type of heat-stable DNA dependent DNA polymerases used). However in extreme cases, the successful PCR amplification of regions of DNA over 35 kilobases in length have been reported using special heatstable DNA dependent DNA polymerases combinations [Hu et al., 2002; Davies and Gray, 2002] . In principle, DNA from all types of viruses, cells (plant, animal and bacterial), or tissues (lung, brain, etc.) may be amplified using nucleic acid extraction techniques coupled to PCR amplification.

As defined in the "central dogma of molecular biology", the flow of genetic information in vivo proceeds from DNA to RNA (via transcription) and then finally to protein (via translation). However, since the central dogma was formulated, scientific discoveries have shown that the flow of genetic information may occur in the reverse direction, i.e. from RNA to DNA (reverse transcription). This process of reverse transcription occurs during the replication cycle of certain virus families (Retroviridae, Hepadnaviridae and Caulimoviridae), and the relevant enzymes (reverse transcriptases) have been isolated and utilized to generate DNA from RNA molecules in specially adapted PCR protocols. This special form of PCR is known as reverse transcriptase-or reverse transcription-PCR (often shortened to RT-PCR), and allows the detection and quantification of several types of RNA molecules, including messenger RNA (mRNA) from both prokaryotic and eukaryotic cells, as well as RNA-based genomes, e.g. Coronaviruses and Picornaviruses. Several non-PCR-based amplification methods have also been developed which do not utilise reverse transcriptase enzymes and hence do not make use of an intermediate DNA template. These techniques amplify RNA (and not double stranded DNA as is the case with PCR) using an initial RNA template.

During in vivo DNA replication, cell associated proteins (including DNAa, DNAb, DNAc, single stranded binding proteins, helicases and gyrases, ligase and a variety of polymerase subunit proteins), all act in concert to uncoil the stable a-helical DNA structure, break the hydrogen bonds between the purine and pyrimidine bases, and expose a DNA replication origin [Nasheuer et al., 2002] . In contrast, DNA required for PCR amplification is separated from its chaperone proteins using chemical and/or enzymatic extraction methods, with the DNA then being separated into single strands via thermal disassociation, i.e. via incubation at approximately 94°C for 30 seconds to 5 minutes, which causes breaking of the hydrogen bonds between the complementary base pairs present on opposite strands. This method is not available to living cells as proteins are rapidly denatured at 94°C and cellular integrity irretrievably breaks down. In fact, the melting temperature (T m ) of a DNA molecule is defined as the temperature at which half of it's constitutive bases are no longer paired to their complementary partner on the opposite strand. For most natural species of DNA, the melting temperature lies somewhere between 70°C and 100°C, with the actual T m being dependent on both the length of the DNA molecule to be melted and on the base composition of the strands. Moreover, there exists a linear relationship between the percentage of guanosine and cytosine bases present in the DNA strands (referred to as the "GC content") and the melting temperature of that particular DNA helix, with GC-rich DNA melting at a higher temperature than adenosine and thymidine rich (AT-rich) DNA. Because of the fact that DNA melting curves are relatively steep, temperatures of T m +5°C and T m −5°C above or below the calculated T m value will result in the double helix being completely denatured or completely intact, respectively.

Several other factors may also facilitate DNA helix destabilization, all of them operating by destabilizing the interaction between the various complementary DNA base moieties. Extreme pH values, denaturants such as formamide or urea and the overall salt concentration are important parameters in this respect (Rauch et al., 2000) . Essentially, the addition of these reagents shifts the DNA melting curve to the left, with the result that the absolute T m for the double stranded DNA value decreases. This decrease in T m may be tens of degrees centigrade dependent on the factor included in the reaction mix. Conversely, several compounds actually stabilize the DNA double helix, including magnesium ions and elevated salt concentrations, which help neutralize the triple-negative phosphate charges on the opposing DNA strands in the duplex (therefore limiting electrostatic repulsion), as well as (partially) neutralizing the effect that the water dipole has on DNA hydrogen bonding. The heating procedure used to melt DNA during the PCR process is a simple and reliable method for ensuring that DNA strand separation occurs and that DNA binding sites for specifically designed PCR primers are exposed. However, at DNA melting temperatures, PCR primers also remain disassociated from the target DNA, and effective binding of the PCR primers to the target DNA can only take place at a reduced temperature when the thermal energy is low enough to allow complementary base pairing. In most applications (random amplification of polymorphic DNA or RAPD excepted), PCR primers are designed to specifically bind to known sequences of target DNA and to anneal to the melted target DNA at a temperature of between 45°C and 65°C (the "annealing" temperature). Of course, at this temperature the target DNA also re-anneals to its complementary strand. However, the excess concentration of primers added to PCR mixes ensures that binding of at least some PCR primers will occur during each thermocycle (dependent of course on the presence of primer-complementary sequences in the target DNA).

DNA dependent DNA polymerase is an essential component of both in vivo DNA replication and the PCR process ( Fig. 2.2) , though the DNA dependent DNA polymerases found in the vast majority of organisms are heat sensitive (thermolabile), one of the major stumbling blocks hindering the initial success of PCR. However, the discovery of thermophilic organisms and the subsequent isolation of thermostable DNA dependent DNA polymerases from these organisms heralded a new chapter in PCR amplification technology [Saiki et al., 1988] , such that successive cycles of heating and cooling (necessary for target DNA melting and primer annealing during PCR thermocycling) no longer resulted in the concomitant denaturation of the thermolabile DNA dependent DNA polymerase enzyme. The discovery of these thermostable enzymes allowed the whole PCR process to become far more convenient, less time consuming and more user-friendly for laboratory personnel. The first thermostable DNA dependent DNA polymerase enzyme to be widely used in PCR (Taq polymerase) was derived from the hot spring-dwelling bacterium Thermus aquaticus, though many other commercially available non-Taq thermostable DNA dependent DNA polymerase enzymes are now available on the market, e.g. Pfu from Pyrococcus furiosus, Vent from Thermococcus litoralis, etc. The advantages and disadvantages of some of these different thermostable polymerases with respect to PCR thermocycling are described more fully in Chapter 7. DNA dependent DNA polymerases per se are actually incapable of performing DNA synthesis from a purely single stranded piece of DNA and require an additional shorter "priming" oligonucleotide to initiate DNA replication. In in vivo DNA replication, an RNA oligonucleotide (generated by a DNA dependent RNA polymerase called primase) acts as the primer for DNA replication. This primase enzyme synthesizes a short RNA molecule (of approximately ten nucleotides), which is complementary to the DNA region to be replicated, and once hybridized acts as a "primer" for DNA replication by cellular DNA dependent DNA polymerase. The primase enzyme itself does not require a primer in order to generate this short RNA oligonucleotide. In contrast, a typical PCR uses two specifically designed and synthetically synthesized 15-25 base pair long oligonucleotides to act as primers for the DNA dependent DNA polymerase enzyme (see Fig. 1 .1, Chapter 1). PCR primers are designed such that one primer is complementary to the sense (coding) template strand and one primer complementary to the antisense (non-coding) template strand, and as such are the major components, in determining PCR specificity. Further, the two PCR primers determine the length of the DNA region to be amplified and facilitate the exponential (two-fold or doubling) of amplification products during each PCR cycle.

In vivo DNA replication and PCR not only require specific primers (RNA for in vivo replication, DNA for PCR amplification) and a (thermostable) DNA polymerase, but also several other factors. The most essential of these factors are the building Fig. 2. 2 Schematic outline of the structure of the DNA dependent DNA polymerase I holoenzyme from Escherichia coli. The amino terminal domain has exonuclease activity, which is able to remove RNA primers and thymidine dimers and to induce strand displacement. The deletion of this domain generates an enzyme that is deficient in 5¢-to 3¢-exonuclease activity (Klenow fragment). In contrast, the central domain exhibits 3¢-to 5¢-exonuclease activity, and the carboxy terminal domain is involved in the binding of DNA and co-factors such as magnesium ions and dNTPs. Taq blocks or free deoxynucleotide triphosphate molecules that are incorporated into the growing DNA chain by the DNA dependent DNA polymerase. These compounds can be acquired in vivo as nutrients, but are generally synthesized via complex pathways involving the reduction of ribonucleotide diphosphates and addition of a further phosphate group by a kinase enzyme. The four major deoxynucleotides required for DNA synthesis (adenosine-, guanosine-, thymidine-and cytosinetriphosphate) contain an energy-rich triphosphate moiety, which is utilized by the DNA polymerase to catalyze a phosphodiester link between the 3′-hydroxy terminus of the primer (or previously added deoxynucleotide triphosphate) and the newly acquired deoxynucleotide triphosphate on the growing DNA strand.

Hydrogen bonding between complementary nucleotides on adjacent strands then completes the double stranded primary structure of the DNA molecule. The mechanism by which deoxyribonucleotide triphosphates (dNTPs) are added to the growing DNA chain is identical for both in vivo replication and PCR amplification processes, though the concentration of dNTPs in in vitro amplification reactions may be easily manipulated to artificially high levels. As well as deoxyribonucleotides, successful in vivo DNA synthesis and amplification requires a large number of chemical components such as (1) the bivalent metal ions magnesium (a cofactor for the DNA polymerase enzyme) and manganese and (2) simple chemicals such as sodium chloride, etc. which help to maintain the correct pH of the reaction or allow for the synthesis of new DNA with a complex secondary structure (see Chapters 4-7 and Wilson et al., [2002] for a more detailed discussion).

Long-range PCR

Replicating by the clock

Long PCR based amplification of the entire mitochondrial genome from single parasitic nematodes

DNA replication in the third domain of life

Initiation of eukaryotic DNA replication: regulation and mechanisms

Segregating sister genomes: the molecular biology of chromosome separation

The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase

Functions of eukaryotic DNA polymerases

A theoretical investigation of cell cycle effects and interspecies radiosensitivities

Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA