The Hershey–Chase experiments were a series of experiments conducted in 1952[1] by Alfred Hershey and Martha Chase that helped to confirm that DNA is the genetic material. While DNA had been known to biologists since 1869,[2] many scientists still assumed at the time that proteins carried the information for inheritance because DNA appeared simpler than proteins. In their experiments, Hershey and Chase showed that when bacteriophages, which are composed of DNA and protein, infect bacteria, their DNA enters the host bacterial cell, but most of their protein does not. Although the results were not conclusive, and Hershey and Chase were cautious in their interpretation, previous, contemporaneous and subsequent discoveries all served to prove that DNA is the hereditary material. Knowledge of DNA gained from these discoveries has applications in forensics, crime investigation and genealogy.[clarification needed]
Hershey shared the 1969 Nobel Prize in Physiology or Medicine with Max Delbrück and Salvador Luria for their “discoveries concerning the genetic structure of viruses
DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.
In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bidirectional from the origin. A number of proteins are associated with the replication fork which helps in terms of the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new DNA by adding complementary nucleotides to the template strand.
DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.
DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.
Mismatch repair is strand-specific. During DNA synthesis the newly synthesised (daughter) strand will commonly include errors. In order to begin repair, the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental). In gram-negative bacteria, transient hemimethylation distinguishes the strands (the parental is methylated and daughter is not). However, in other prokaryotes and eukaryotes, the exact mechanism is not clear. It is suspected that, in eukaryotes, newly synthesized lagging-strand DNA transiently contains nicks (before being sealed by DNA ligase) and provides a signal that directs mismatch proofreading systems to the appropriate strand. This implies that these nicks must be present in the leading strand, and evidence for this has recently been found.[3] Recent work [4] has shown that nicks are sites for RFC-dependent loading of the replication sliding clamp PCNA, in an orientation-specific manner, such that one face of the donut-shape protein is juxtaposed toward the 3'-OH end at the nick. Oriented PCNA then directs the action of the MutLalpha endonuclease to one strand in the presence of a mismatch and MutSalpha or MutSbeta.
Any mutational event that disrupts the superhelical structure of DNA carries with it the potential to compromise the genetic stability of a cell. The fact that the damage detection and repair systems are as complex as the replication machinery itself highlights the importance evolution has attached to DNA fidelity.
Examples of mismatched bases include a G/T or A/C pairing (see DNA repair). Mismatches are commonly due to tautomerization of bases during G2. The damage is repaired by recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide. The removal process involves more than just the mismatched nucleotide itself. A few or up to thousands of base pairs of the newly synthesized DNA strand can be removed.
DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.
Messenger RNA (mRNA) is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. The existence of mRNA was first suggested by Jacques Monod and François Jacob. Following transcription of primary transcript mRNA (known as pre-mRNA) by RNA polymerase, processed, mature mRNA is translated into a polymer of amino acids: a protein, as summarized in the central dogma of molecular biology.
As in DNA, mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis. This process of translation of codons into amino acids requires two other types of RNA: Transfer RNA (tRNA), that mediates recognition of the codon and provides the corresponding amino acid, and ribosomal RNA (rRNA), that is the central component of the ribosome's protein-manufacturing machinery.
The polymerase chain reaction (PCR) is a biomedical technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, after which the method is named, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.
In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
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PCR principles and procedure
Figure 1a: A thermal cycler for PCR
Figure 1b: An older model three-temperature thermal cycler for PCR
PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for amplification of fragments up to 40 kbp in size.[6]The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses.[7]
A basic PCR set up requires several components and reagents.[8] These components include:
DNA template that contains the DNA region (target) to be amplified.
Two primers that are complementary to the 3' (three prime) ends of each of thesense and anti-sense strand of the DNA target.
Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Bivalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+concentration increases the error rate during DNA synthesis[9]
Monovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.
Procedure[edit]
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Figure below). The cycling is often preceded by a single temperature step at a high temperature (>90 °C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[10]
Initialization step(Only required for DNA polymerases that require heat activation by hot-start PCR.[11]): This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes.
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature needs to be low enough to allow for hybridization of the primer to the strand, but high enough in order for the hybridization to be specific, i.e. the primer should only bind to a perfectly complementary part of the template. If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind. Typically the annealing temperature is about 3–5 °C below the Tm of the primers used. Stable DNA–DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,[12][13] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of 70–74 °C (this is the temperature needed for optimal activity for most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).
PCR stages[edit]
The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.[14]
Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
PCR optimization[edit]
Main article: PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[15][16] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[8] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide, in buffer systems may increase the specificity and yield of PCR.[17] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.[18]
Application of PCR[edit]
Main article: Applications of PCR
Selective DNA isolation[edit]
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.
Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNAtechnologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E. coli) can be rapidly screened by PCR for correct DNA vector constructs.[19] PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms.[citation needed]
Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.
Amplification and quantification of DNA[edit]
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ADNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptianmummies to the identification of a Russian tsar and the body of English king Richard III.[20]
Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels ofgene expression. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
See also: Use of DNA in forensic entomology
PCR in diagnosis of diseases
PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods.
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or virusesPCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead time in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).
Variations on the basic PCR technique
:
Allele-specific PCR: a diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with SNPs) (single-base differences in a patient). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated). PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.See SNP genotyping for more information.
Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.
Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencingand hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR.
Digital PCR (dPCR): used to measure the quantity of a target DNA sequence in a DNA sample. The DNA sample is highly diluted so that after running many PCRs in parallel, some of them will not receive a single molecule of the target DNA. The target DNA concentration is calculated using the proportion of negative outcomes. Hence the name 'digital PCR'.
Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
Hot start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95 °C) before adding the polymerase Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an ] or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome. In silico PCR was proposed as an educational tool for molecular biology.
Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.
Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.
Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis.
Nanoparticle-Assisted PCR (nanoPCR): In recent years, it has been reported that some nanoparticles (NPs) can enhance the efficiency of PCR (thus being called nanoPCR), and some even perform better than the original PCR enhancers. It was also found that quantum dots (QDs) can improve PCR specificity and efficiency. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are efficient in enhancing the amplification of long PCR. Carbon nanopowder (CNP) was reported be able to improve the efficiency of repeated PCR and long PCR. ZnO, TiO2, and Ag NPs were also found to increase PCR yield. Importantly, already known data has indicated that non-metallic NPs retained acceptable amplification fidelity. Given that many NPs are capable of enhancing PCR efficiency, it is clear that there is likely to be great potential for nanoPCR technology improvements and product development.
Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR or Splicing by overlap extension (SOEing) : a genetic engineering technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs. It can also introduce deletions, insertions or point mutations into a DNA sequence.
PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.
quantitative PCR (qPCR): used to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. quantitative PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative PCR has a very high degree of precision. Quantitative PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (real-time PCR) but this abbreviation should be used only for reverse transcription PCR. qPCR is the appropriate contractions for quantitative PCR (real-time PCR).
Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNAinto cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).
Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR(where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).
Suicide PCR: typically used in paleogenetics or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority. It was originally described in a study to verify the presence of the microbe Yersinia pestis in dental samples obtained from 14th Century graves of people supposedly killed by plague during the medieval Black Death epidemic.[45] The method prescribes the use of any primer combination only once in a PCR (hence the term "suicide"), which should never have been used in any positive control PCR reaction, and the primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.
Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.[46]
Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5 °C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5 °C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer — which can lead to artefactual 'noise')by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),5'RACE LaNe[ and 3'RACE LaNe.
The polymerase chain reaction (PCR) is a biomedical technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, after which the method is named, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.
In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
DNA condensation refers to the process of compacting DNA molecules in vitro or in vivo.Mechanistic details of DNA packing are essential for its functioning in the process of gene regulation in living systems. Condensed DNA often has surprising properties, which one would not predict from classical concepts of dilute solutions. Therefore DNA condensation in vitro serves as a model system for many processes of physics, biochemistry and biology.In addition, DNA condensation has many potential applications in medicine and biotechnology.
DNA diameter is about 2 nm, while the length of a stretched single molecule may be up to several dozens of centimetres depending on the organism. Many features of the DNA double helix contribute to its large stiffness, including the mechanical properties of the sugar-phosphate backbone, electrostatic repulsion between phosphates (DNA bears on average one elementary negative charge per each 0.17 nm of the double helix), stacking interactions between the bases of each individual strand, and strand-strand interactions. DNA is one of the stiffest natural polymers, yet it is also one of the longest molecules. This means that at large distances DNA can be considered as a flexible rope, and on a short scale as a stiff rod. Like a garden hose, unpacked DNA would randomly occupy a much larger volume than when it is orderly packed. Mathematically, for a non-interacting flexible chain randomly diffusing in 3D, the end-to-end distance would scale as a square root of the polymer length. For real polymers such as DNA this gives only very rough estimate; what is important, is that the space available for the DNA in vivo is much smaller than the space that it would occupy in the case of a free diffusion in the solution. In order to cope with the volume constraints, DNA has a striking property to pack itself in the appropriate solution conditions with the help of ions and other molecules. Usually, DNA condensation is defined as "the collapse of extended DNA chains into compact, orderly particles containing only one or a few molecules". This definition applies to many situations in vitro and is also close to the definition of DNA condensation in bacteria as "adoption of relatively concentrated, compact state occupying a fraction of the volume available".In eukaryotes, the DNA size and the number of other participating players are much larger, and a DNA molecule forms millions of ordered nucleoprotein particles, the nucleosomes, which is just the first of many levels of DNA packing.
Meiosis Listeni/maɪˈoʊsɨs/ is a specialized type of cell division which reduces the chromosome number by half. This process occurs in all sexually reproducing eukaryotes (both single-celled and multicellular) including animals, plants, and fungi.
In meiosis, DNA replication is followed by two rounds of cell division to produce four daughter cells with half the number of chromosomes as the original parent cell. The two meiotic divisions are known as meiosis I and meiosis II. Before meiosis begins, during S phase of the cell cycle, the DNA of each chromosome is replicated so that it consists of two identical sister chromatids attached at a centromere. In meiosis I, homologous chromosomes pair with each other and can exchange genetic material in a process called crossing over. The homologous chromosomes are then pulled apart into two new separate daughter cells, each containing half the number of chromosomes as the parent cell. At the end of meiosis I, sister chromatids remain attached and may differ from one another if crossing-over occurred. In meiosis II, the two cells produced during meiosis I divide again. During this division, sister chromatids detach from one another and are separated into four total daughter cells. These cells can mature into gametes, spores, pollen, and other reproductive cells.
Because the number of chromosomes is halved during meiosis, it allows gametes to fuse (i.e. fertilization) to form a zygote containing a mixture of paternal and maternal chromosomes. Thus, meiosis and fertilization facilitate sexual reproduction with successive generations maintaining the same number of chromosomes. For example, a typical diploid human cell contains 23 pairs of chromosomes (46 total, half of maternal origin and half of paternal origin). Meiosis produces haploid gametes with one set of 23 chromosomes. When two gametes (an egg and a sperm) fuse, the resulting zygote is once again diploid, with the mother and father each contributing 23 chromosomes.
The basic repeating structural (and functional) unit of chromatin is the nucleosome, which contains nine histone proteins and about 166 base pairs of DNA (Van Holde, 1988; Wolffe, 1999). The observation by electron microscopists that chromatin appeared similar to beads on a string provided an early clue that nucleosomes exist (Olins and Olins, 1974; Woodcock et al., 1976). Another clue came from chemically cross-linking (i.e., joining) histones in chromatin (Thomas & Kornberg, 1975). This experiment demonstrated that H2A, H2B, H3, and H4 form a discrete protein octamer, which is fully consistent with the presence of a repeating histone-containing unit in the chromatin fiber.
Today, researchers know that nucleosomes are structured as follows: Two each of the histones H2A, H2B, H3, and H4 come together to form a histone octamer, which binds and wraps about 1.7 turns of DNA, or about 146 base pairs. The addition of one H1 protein wraps another 20 base pairs, resulting in two full turns around the octamer (Figure 1). Obviously, 166 base pairs are not very much, as each chromosome contains over 100 million base pairs of DNA on average. Therefore, every chromosome contains hundreds of thousands of nucleosomes, and these nucleosomes are joined by the DNA that runs between them (an average of about 20 base pairs). This joining DNA is referred to as linker DNA. Each chromosome is thus a long chain of nucleosomes, which gives the appearance of a string of beads when viewed using an electron microscope (Figure 2; Olins & Olins, 1974, 2003).
A digital model shows the atomic structure of a nucleosome bound to a DNA molecule, from a top-down perspective and in profile against a black background. In the top-down image, at left, a green and brown DNA double-helix forms a closed circle around a complex of blue, green, yellow, and red coiled ribbons. Each ribbon represents a histone protein. In the profile image, at right, the double-helix is wound around the histone proteins in an upside down U-shape.
View Full-Size ImageFigure 3
The amount of DNA per nucleosome was determined by treating chromatin with an enzyme that cuts DNA (such enzymes are called DNases). One such enzyme, micrococcal nuclease (MNase), has the important property of preferentially cutting the linker DNA between nucleosomes well before it cuts the DNA that is wrapped around octamers. By regulating the amount of cutting that occurs after application of MNase, it is possible to stop the reaction before every linker DNA has been cleaved. At this point, the treated chromatin will consist of mononucleosomes, dinucleosomes (connected by linker DNA), trinucleosomes, and so forth (Hewish and Burgoyne, 1973).If DNA from MNase-treated chromatin is then separated on a gel, a number of bands will appear, each having a length that is a multiple of mononucleosomal DNA (Noll, 1974). The simplest explanation for this observation is that chromatin possesses a fundamental repeating structure. When this was considered together with data from electron microscopy and chemical cross-linking of histones, the "subunit theory" of chromatin (Kornberg, 1974; Van Holde et al., 1974) was adopted. The subunits were later named nucleosomes (Oudet et al., 1975) and were eventually crystallized (Luger et al., 1997). The model of the nucleosome that crystallographers constructed from their data is shown in Figure 3. Phosphodiester backbones of the DNA double helix are shown in brown and turquoise, while histone proteins are shown in blue (H3), green (H4), yellow (H2A), and red (H2B). Note that only eukaryotes (i.e., organisms with a nucleus and nuclear envelope) have nucleosomes. Prokaryotes, such as bacteria, do not.
Chromatin Is Coiled into Higher-Order Structures
A black-and-white electron micrograph shows a 30-nanometer chromatin fiber. The fiber appears as a black, vertical, elongated tube against a light grey background. It starts in the upper left, curves toward the right, and then curves back and ends at the center bottom. Other short black tubes appear in the upper right and left middle and bottom.
View Full-Size ImageFigure 4
The packaging of DNA into nucleosomes shortens the fiber length about sevenfold. In other words, a piece of DNA that is 1 meter long will become a "string-of-beads" chromatin fiber just 14 centimeters (about 6 inches) long. Despite this shortening, a half-foot of chromatin is still much too long to fit into the nucleus, which is typically only 10 to 20 microns in diameter. Therefore, chromatin is further coiled into an even shorter, thicker fiber (Figure 4), termed the "30-nanometer fiber," because it is approximately 30 nanometers in diameter. Over the years, there has been a great deal of speculation concerning the manner in which nucleosomes are folded into 30-nanometer fibers (Woodcock, 2005). Part of the problem lies in the fact that electron microscopy is perhaps the best way to visualize packaging, but individual nucleosomes are hard to discern after the fiber has formed. In addition, it also makes a difference whether observations are made using isolated chromatin fibers or chromatin within whole nuclei. Thus, the 30-nanometer fiber may be highly irregular and not quite the uniform structure depicted in textbooks (Figure 1; Bednar et al., 1998). Interestingly, histone H1 is very important in stabilizing chromatin higher-order structures, and 30-nanometer fibers form most readily when H1 is present.
Processes such as transcription and replication require the two strands of DNA to come apart temporarily, thus allowing polymerases access to the DNA template. However, the presence of nucleosomes and the folding of chromatin into 30-nanometer fibers pose barriers to the enzymes that unwind and copy DNA. It is therefore important for cells to have means of opening up chromatin fibers and/or removing histones transiently to permit transcription and replication to proceed. Generally speaking, there are two major mechanisms by which chromatin is made more accessible:
Histones can be enzymatically modified by the addition of acetyl, methyl, or phosphate groups (Fischle et al., 2005).
Histones can be displaced by chromatin remodeling complexes, thereby exposing underlying DNA sequences to polymerases and other enzymes (Smith & Peterson, 2005).
It is important to remember that these processes are reversible, so modified or remodeled chromatin can be returned to its compact state after transcription and/or replication are complete.
Chromosomes Are Most Compacted During Metaphase
When eukaryotic cells divide, genomic DNA must be equally partitioned into both daughter cells. To accomplish this, the DNA becomes highly compacted into the classic metaphase chromosomes that can be seen with a light microscope. Once a cell has divided, its chromosomes uncoil again.
Comparing the length of metaphase chromosomes to that of naked DNA, the packing ratio of DNA in metaphase chromosomes is approximately 10,000:1 (depending on the chromosome). This can be thought of as akin to taking a rope as long as a football field and compacting it down to less than half an inch. This level of compaction is achieved by repeatedly folding chromatin fibers into a hierarchy of multiple loops and coils (Figure 1). Exactly how this is accomplished is unclear, but the phosphorylation of histone H1 may play a role. Indeed, this is just one area of DNA packaging that researchers will continue to explore in the years to come.
The Mechanism of initiation Prokaryotic DNA replication
Initiation
The initiation of DNA replication is mediated by DnaA, a protein that binds to a region of the origin known as the DnaA box. In E. coli, there are 4 DnaA boxes, each of which contains a highly conserved 9 bp consensus sequence 5' - TTATCCACA - 3'. Binding of DnaA to this region causes it to become negatively supercoiled. Following this, a region of OriC upstream of the DnaA boxes (known as DnaB boxes) become melted. There are three of these regions, and each is 13 bp long, and AT-rich (which facilitates melting because less energy is required to break the two hydrogen bonds that form between A and T nucleotides). This region has the consensus sequence 5' - GATCTNTTNTTTT - 3. Melting of the DnaB boxes requires ATP (which is hydrolyzed by DnaA). Following melting, DnaA recruits a hexameric helicase (six DnaB proteins) to opposite ends of the melted DNA. This is where the replication fork will form. Recruitment of helicase requires six DnaC proteins, each of which is attached to one subunit of helicase. Once this complex is formed, an additional four DnaA proteins bind to the original four DnaA proteins to form four DnaA dimers. DnaC is then released, and the prepriming complex is complete. In order for DNA replication to continue, SSB protein is needed to prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing, and DNA gyrase is needed to relieve the stress (by creating negative supercoils) created by the action of DnaB helicase. The unwinding of DNA by DnaB helicase allows for primase (DnaG) and RNA polymerase to prime each DNA template so that DNA synthesis can initiate.
DNA Polymerase I (or Pol I) is an enzyme that participates in the process of DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and, indeed, the first known of any kind of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli form of the enzyme is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations.
Pol I possesses four enzymatic activities:
A 5' -> 3' (forward) DNA-Dependent DNA polymerase activity, requiring a 3' primer site and a template strand
A 3' -> 5' (reverse) exonuclease activity that mediates proofreading
A 5' -> 3' (forward) exonuclease activity mediating nick translation during DNA repair.
A 5' -> 3' (forward) RNA-Dependent DNA polymerase activity. Pol I operates on RNA templates with considerably lower efficiency (0.1–0.4%) than it does DNA templates, and this activity is probably of only limited biological significance.
In the replication process, RNAse H removes the RNA primer (created by Primase) from the lagging strand and then Polymerase I fills in the necessary nucleotides between the Okazaki fragments (see DNA replication) in 5' -> 3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme - it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template. DNA Ligase then joins the various fragments together into a continuous strand of DNA.
Despite its early characterisation, it quickly became apparent that Polymerase I was not the enzyme responsible for most DNA synthesis — DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, while the rate of base pair synthesis by Polymerase I averages only between 10 and 20 nucleotides/second. Moreover, its cellular abundance of approximately 400 molecules per cell did not correlate with the fact that there are typically only two replication forks in E. coli. Moreover, it is insufficiently processive to copy an entire genome, as it falls off after incorporating only 25-50 nucleotides. Its role in replication was proven when, in 1969, John Cairns isolated a viable Polymerase I mutant that lacked the polymerase activity. Cairns' lab assistant Paula De Lucia created thousands of cell free extracts from E.coli colonies and assayed them for DNA-polymerase activity. The 3,478th clone contained the polA mutant, which was named by Cairns to credit "Paula" [De Lucia]. It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.
