Polymerase Chain Reaction (PCR) Steps

Polymerase Chain Reaction (PCR) Steps We owe the discovery of the polymerase chain reaction to Kary B Mullis in the year 1983. He was the actual proponent of PCR. Few people are aware that in 1971, Kleppe and the Nobel laureate Gobind Khorana published studies including a description of techniques that are now known to be the basis for nucleic acid replication. However, it is unfortunate that Kleppe and Khorana were ahead of their times. Oligonucleotide synthesis wasnt as simple as it is today; genes had not been sequenced and the idea of thermostable DNA polymerases had not been described. Hence, the credit for discovering the PCR remains with Kary Mullis. The Polymerase Chain Reaction is essentially a cell-free method of DNA and RNA cloning. The DNA or RNA is isolated from the cell and replicated upto a million times. At the end, what you get is a greatly amplified fragment of DNA. The PCR is quick, reliable and sensitive and its variations have made it the basis of genetic testing. WHAT KARY B MULLIS SAYS ABOUT HOW HE DISCOVERED THE POLYMERASE CHAIN REACTION I was just driving and thinking about ideas and suddenly I saw it. I saw the polymerase chain reaction as clear as if it were up on a blackboard inside my head, so I pulled over and started scribbling. A chemist friend of his was asleep in the car. Mullis says that Jennifer objected groggily to the delay and the light, but I exclaimed I had discovered something fantastic. Unimpressed, she went back to sleep. Mullis kept scribbling calculations, right there in the car. He convinced the small California biotech company, Cetus, he was working for at that time, that he was up to something big. They finally listened. They sold the patent of PCR to Hoffman-LaRoche for a staggering $300 million the maximum amount of money ever paid for a patent. Mullis meanwhile received a $10,000 bonus. BASIS OF THE METHOD The purpose of PCR is to generate a huge number of copies of a segment of DNA, which could be a gene, a portion of a gene, or an intronic region. There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can either heat or cool the tubes containing the reaction mixture, as required, in a very short period of time. There are three major steps in a PCR, which are repeated for 30 or 40 cycles. DenaturationDuring this process, the double stranded DNA melts and opens to form single stranded DNA. All enzymatic reactions, such as those carried over from a previous cycle, stop. This will be explained in the next paragraph. The temperature for denaturation is not fixed but it usually occurs at about 95 °C. It is important to realize that the denaturation temperature is largely dependent on G:C (guanine:cytosine) content of the DNA fragment to be analyzed. This is reasonable when one considers that the G:C bond is a triple hydrogen bond and the AT bond is a double bond. Logic dictates that a triple bond should be 1.5 times harder to break than a double bond. Therefore, when the segment of DNA to be analyzed has a very high G:C content, the denaturation temperature can reach even upto 99 °C. AnnealingThis requires temperatures lower than those required for denaturation. In this process, the primers anneal to that very specific segment of DNA that is to be amplified. The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of what is now double stranded DNA (template and primer); the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. ExtensionThis is done at 72 °C. This is the ideal temperature for working with polymerase. The primers, which are complementary to the template, already have a strong ionic attraction to the template. This force is stronger than the forces breaking these attractions i.e. the high temperature. Primers that are on positions with no exact match (non complementary) get loose again (because of the higher temperature) and dont give an extension of the fragment. The nucleotide bases are added from the 5 end to the 3 end. The phosphate group of the dNTPs is coupled with the hydroxyl group of the extending DNA strand. The extension time depends on two factors; the type of polymerase used and the length of the DNA fragment to be amplified. Usually, Taq polymerase adds dNTPs at the rate of about 1000 bases per minute. It is important to realize that each component of the PCR including the input DNA, the oligonucleotide primers, the thermostable polymerase, the buffer and the cycling parameters has a profound impact on the sensitivity, specificity and fidelity of the reaction. The three steps of the first cycle are shown, that is, denaturation, annealing and extension. At the end of the first cycle, two strands have been synthesized. At the end of the second cycle, four strands have been synthesized (the three steps of the cycle have not been shown). At the end of the third cycle, eight strands have been synthesized. The number of strands increases exponentially with each cycle. Nuggets The Polymerase Chain Reaction is essentially a cell-free method of cloning DNA and RNA. There are three steps involved in every cycle; these are denaturation, annealing and extension. At the end of each cycle, the DNA doubles. Therefore, theoretically, if there are n cycles in a reaction, the number of DNA fragments at the end of the reaction will be 2n. COMPONENTS OF THE POLYMERASE CHAIN REACTION The components that are essential for a successful PCR are elaborated here. TEMPLATE DNA This is that portion of the DNA/gene that is to be amplified. Usually the concentration is  100 ng genomic DNA per PCR reaction. However, this can vary depending on the target gene concentration and the source of DNA. The PCR reaction is inherently sensitive. It is not necessary for the template DNA to be abundant or highly purified. Higher amounts of template DNA can increase the yield of nonspecific PCR products, but if the fidelity of the reaction is crucial, one should limit both template DNA quantities as well as the number of PCR cycles. DNA in solution may contain a large number of contaminants. These contaminants may inhibit the PCR. Some of these reagents are phenol, EDTA, and proteinase K, which can inhibit Taq DNA polymerase. However, isopropanol precipitation of DNA and washing of DNA pellets with 70% ethanol is usually effective in removing traces of contaminants from the DNA sample. Effects of Fixation This is of particular interest to the pathologist since he has to deal with formalin fixed tissue. DNA extracted from fresh tissue or cell suspensions forms an optimal template for PCR. The tissue is best stored at -70 °C at which the nucleic acids can be stored indefinitely. A temperature of -20 °C is sufficient to preserve the DNA for several months and at 4 °C, the DNA can be stored for several weeks. At room temperature, the DNA has been successfully stored for hours to days; however, mitochondrial DNA is very sensitive to temperature and may degrade in thawed tissues. DNA extracted from fixed tissue has been used successfully for PCR. The type of fixative and the duration of fixation are of critical importance. Non crosslinking fixatives like ethanol provide the best DNA. Formaldehyde is variable in its DNA yield. Carnoys, Zenkers and Bouins are poor fixatives as far as DNA preservation is concerned. Not surprisingly, formaldehyde is the fixative which has been evaluated the most, because it is more commonly used worldwide. The studies have demonstrated that a successful PCR depends on the protocol to extract the DNA and the length of fixation. Formaldehyde reacts with DNA and proteins to form labile hydroxymethyl intermediates which give rise to a mixture of end products which include DNA-DNA and DNA-protein adducts. Purification of DNA from formalin fixed tissue, therefore, includes heating to reverse the hydroxymethyl additions and treatment with a proteinase to hydrolyze the covalently linked proteins. However, there is no way to reverse the DNA-DNA links and these links inhibit the DNA polymerases. This accounts for the low PCR yield which is seen with formalin fixed tissue. Usually, the PCR reaction with formalin fixed DNA as a template yields products which are not more than 600 bp in size. Nuggets Template DNA is required in a concentration of 100ng for each PCR reaction. Contaminants in DNA may inhibit the reaction. Fixation of tissues provides DNA which is not as good as DNA obtained from fresh/ frozen tissues. Different fixatives give different DNA yields. Alcohol is the best fixative and Carnoys, Zenkers and Bouins are poor fixatives as far as DNA preservation is concerned. Formalin is intermediate in DNA yield. Purification of DNA from formalin fixed tissue involves heating to reverse the attachment of hydroxymethyl intermediates and treatment with a proteinase to hydrolyze the covalently linked proteins. The DNA obtained after fixation can be used for reactions in which the PCR product is not more than 600 bp. PCR BUFFER The purpose of using buffers in PCR is to provide optimum pH and potassium ion concentration for the DNA polymerase enzyme (usually obtained from bacteria Thermus aquaticus) to function. Most buffers are available in a 10X concentration and require dilution before use. Although most protocols recommend the final buffer concentration of 1X, a concentration of 1.5X might result in increased PCR product yield. The PCR buffer contains many components. Some important ones are discussed here: Divalent and monovalent cations These are required by all thermostable DNA polymerases. Mg2+ is the divalent cation that is usually present in most of the PCR buffers. Some polymerases also work with buffers containing Mn2+. Calcium containing buffers are ineffective and therefore, rarely used. Buffers can be divided into first and second generation buffers on the basis of their ionic component. The second generation buffers, as opposed to first generation buffers, also contain (NH4)2SO4 and permit consistent PCR product yield and specificity over a wide range of magnesium concentration (1.0 to 4.0 mM MgCl2). The overall specificity and yield of PCR products is better with second generation buffers, as compared with first generation PCR buffers. Buffers also contain KCl. Salts like KCl and NaCl may help to facilitate primer annealing, but concentration of 50 mM will inhibit Taq polymerase activity. Interactions between K+ and NH4+ allow specific primer hybridization over a broad ran ge of temperatures. Magnesium is one of the most important components of the buffer. Mg2+ ions form a soluble complex with dNTPs which is essential for dNTP incorporation; they also stimulate polymerase activity and influence the annealing efficiency of primer to template DNA. The concentration of MgCl2 can have a dramatic effect on the specificity and yield of PCR products. Optimal concentration of MgCl2 is between 1.0 to 1.5 mM for most reactions. Low MgCl2 concentration helps to eliminate non-specific priming and formation of background PCR products. This is desirable when fidelity of DNA synthesis is critical. At the same time, however, too few Mg2+ ions can result in low yield of PCR products. High MgCl2 concentration helps to stabilize interaction of the primers with their intended template, but can also result in nonspecific binding and formation of non specific PCR products. It is important to be aware that many PCR buffers (often sold in 10X stocks) already contain some amo unt of MgCl2. Therefore, the addition of further amounts must be carefully monitored. In the best possible scenario, the PCR would work well with the amount of Mg2+ already present in the buffer solution. However, if this does not occur, it is necessary to standardize the amount of Mg2+ in the reaction mix. This can be difficult because the dNTPs and the oligonucleotide primers bind to Mg2+. Therefore, the molar concentration of Mg2+ must exceed the molar concentration of the phosphate groups contributed by dNTPs and the primers. As a rule of thumb, the magnesium concentration in the reaction mixture is generally 0.5 to 2.5 mM greater than the concentration of dNTPs. The optimal concentration of Mg2+ should, therefore, be standardized for each reaction. Tris-Cl The concentration of tris-Cl is adjusted so that the pH of the reaction mixture is maintained between 8.3 and 8.8 at room temperature. In standard PCR reactions, it is usually present in a concentration of 10mM. When incubated at 72 °C which is the temperature for extension, the pH of the reaction mixture falls by more than a full unit, producing a buffer whose pH is 7.2. Other components Some buffers also contain components like BSA (Bovine serum albumin) and DMSO (dimethyl sulphoxide). BSA reduces the amount of template sticking to the side of the tube, making it available for amplification and reducing the risk of primer dimer. Primer dimers are products obtained when the primers anneal to each other instead to to the template DNA. DMSO has been shown to facilitate DNA strand separation (in GC rich difficult secondary structures) because it disrupts base pairing and has been shown to improve PCR efficiency. In effect, it is wise not to tamper with the buffer provided with the Taq polymerase. The buffer is usually standardized for the vial of Taq and there is no need to add additional MgCl2 or stabilizers like DMSO and BSA. However, some Taq buffers come with the buffer in one vial and MgCl2 in a separate vial. Under such circumstances, it is advisable to start with 1 µL of MgCl2 and increase its concentration in aliquots of 0.5  µL, if the initial reaction fails. Nuggets The PCR buffer contains divalent and monovalent cations, Tris Cl and other components. The PCR buffer is used to give the correct pH and potassium concentration for the DNA polymerase to function. The most common divalent ion used is magnesium in the form of MgCl2. MgCl2 concentration is vital for PCR. Tris Cl is used to maintain the pH between 8.3 and 8.8 at room temperature. Salts like NaCl and KCl may facilitate primer annealing Other components like BSA and DMSO help to increase the sensitivity and specificity of the reaction. OLIGONUCLEOTIDE PRIMERS What are Oligonucleotide Primers? PCR primers are short fragments of single stranded DNA (17-30 nucleotides in length) that are complementary to DNA sequences that flank the target region of interest. The purpose of PCR primers is to provide a free 3-OH group to which the DNA polymerase can add dNTPs. There are two primers used in the reaction. The forward primer anneals to the DNA minus strand and directs synthesis in a 5 to 3 direction. The sequence of primers is always represented in a 5 to a 3 direction. The reverse primer anneals to the other strand of the DNA. How to design a primer? The predominant goal kept in mind while designing a primer is specificity. Each member of the primer must anneal in a stable fashion to its target sequence in the template DNA. The longer the primer, the higher is its specificity. Unfortunately, the longer the primer, the less likely it is to anneal to a particular sequence in the template DNA. Conversely, if the primer length is small, it is likely to anneal, but its specificity will be poor. A compromise is reached by designing primers between 20 and 25 nucleotides long. Inclusion of less than 17 nucleotides often leads to non specific annealing, while presence of more than 25 nucleotides may not allow annealing to occur at all. Remember that the DNA sequence in the human genome appears to be a random sequence of nucleotides. When designing primers, it is important to calculate the probability that a sequence exactly complementary to a string of nucleotides in the human genome will occur by chance. Several formulae are designed to calculate such probabilities. However, mathematical expressions are not necessarily correct and in this case, the predictions maybe wildly wrong. The distribution of codons is non random with repetitive DNA sequences and gene families. It is advisable to use primers longer than the statistically indicated minimum. It is also advisable to scan DNA databases to check if the proposed sequence occurs only in the desired gene. For a practicing pathologist, it is best not to attempt designing of primers. What a pathologist requires is the primer sequence for an established test. If, for example, a pathologist requires primer sequence for the diagnosis of sickle cell anemia, all he has to do is search the web for papers related to molecular testing of sickle cell anemia. The primer sequences will be provided in the paper. Custom made primers can be commercially synthesized. Several biotechnology companies provide this facility. Before the primers are ordered, it is essential to check that the sequence is correct and that there are no missing nucleotides in the sequence. That is where, BLAST is invaluable. Before the intricacies of the BLAST search are elaborated upon, it is necessary to mention that designing a primer does not depend only on the sequence of nucleotides. Other factors like the GC content and melting point are also important considerations. They will be dealt with later in the chapter. BLAST and its uses BLAST is an acronym for Basic Local Alignment Search Tool. It is an algorithm comparing information about primary biological sequences with a library or database of sequences. A BLAST can be performed for different organisms, but in this book, we will concern ourselves with nucleotide BLAST in humans only. BLAST searches the database for sequences similar to the sequence of interest (the query sequence) by using a 2-step approach. The basic concept is that the higher the number of similar segments between two sequences, and the longer the length of similar segments, the less divergent the sequences are, and therefore, likely to be more genetically related (homologous). Before perfoming a BLAST search the oligonucleotide sequence is first identified. The sequence is fed into the programme. BLAST first searches for short regions of a given length called words (W). It then searches for substrings which are compared to the query sequence. The program then aligns with sequences in the database (target sequences), using a substitution matrix. For every pair of sequences (query and target) that have a word or words in common, BLAST extends the search in both directions to find alignments that score greater (are more similar) than a certain score threshold (S). These alignments are called high scoring pairs or HSPs; the maximal scoring HSPs are called maximum segment pairs (MSPs). The BLAST search as outlined in fig 7.2 shows the results of the search. If we scroll down further, we can see the sequences producing significant alignments. Note that in this BLAST search, there are 49 BLAST hits in the query sequence. In the list shown in figure 7.2, there is a list of hits starting with the best (most similar). To the right of the screen is the E-value. This is the expected number of chance alignments; the lower the E value, the more significant the score. First in the list is the sequence finding itself, which obviously has the best score. To the left is the accession number. This refers to a unique code that identifies a sequence in a database. It is important to know that there is no set cut-off that determines whether a match is significant or similar enough. This must be determined according to the goals of the project. The sequences provided in the figure 7.2 show a significant alignment with Pseudomonas japonica. It shows a high score (bits) and a low E-value. Note that the lower the E value, the greater the likelihood that the sequence is a good match. BLAST output can be delivered in a variety of formats. These formats include HTML, plain text and XML formatting. For the NCBIs web-page, the default format for output is HTML. When performing a BLAST on NCBI (National Centre for Biotechnology Information), the results are displayed in a graphical format showing the following: The hits found A tabular form showing sequence identifiers for the hits with scoring related data Alignments for the sequence of interest and the hits received with corresponding BLAST scores for these. The easiest to read and most informative of these is probably the table. The main idea of BLAST is that there are often high-scoring segment pairs (HSP) in a statistically significant alignment. BLAST searches for these high scoring sequence alignments between the query sequence and the sequences in the database. The speed and relatively good accuracy of BLAST are among the key technical innovations of the BLAST programs. Sequence of events to be followed when performing a BLAST search.: Go to PUBMED (http://www.ncbi.nlm.nih.gov/pubmed/) Scroll down to reach a heading called POPULAR Under POPULAR click on BLAST Click on nucleotide blast Under the heading, enter accession number(s), gi(s), or FASTA sequence(s), type or paste the sequence that you want matched. Click BLAST Wait for the results. Analyse the nucleotide sequence as it appears. Calculation of Melting Temperature The melting temperature or Tm is a measure of stability of the duplex formed by the primer and the complementary target DNA sequence and is an important consideration in primer design. Tm corresponds to the midpoint in transition of DNA from the double stranded to its single stranded form. A higher Tm permits an increased annealing temperature that makes sure that the annealing between the target DNA and the primer is specific. The Tm is dependent on the length of the oligonucleotides and the G+C content of the primer. The formula for calculation of Tm is given in table 7.1. Table 7.1: Formula for calculation of the melting temperature. Length of Primer Tm ( °C) Less than 20 nucleotides long 2(effective length*) 20 to 35 nucleotides long 22 + 1.46(effective length) *Effective length = 2(number of G+C) + number of (A + T) Primers are usually designed to avoid matching repetitive DNA sequences. This includes repeats of a single nucleotide.. The two primers in a PCR reaction are not homologous to each other and their complementarity can lead to formation of spurious amplification artifacts called primer dimers. The 3 end of a primer is most critical for initiating polymerization. The rules for selecting primers in addition to those already mentioned are as follows: The C and G nucleotides should be distributed uniformly throughout the primer and comprise approximately 40% of the bases. More than three G or C nucleotides at the 3-end of the primer should be avoided, as nonspecific priming may occur. The primer should be neither self-complementary nor complementary to any other primer in the reaction mixture, in order to avoid formation of primer-dimer or hairpin-like structure. All possible sites of complementarity between the primer and the template DNA should be noted. The melting temperature of flanking primers should not differ by more than 5 °C. Therefore, the G+C content and length must be chosen accordingly (a higher G+C content means a higher melting temperature). The PCR annealing temperature (TA) should be approximately 5 °C lower than the primer melting temperature. G+C content in each primer should not be more than 60% to avoid formation of internal secondary structures and long stretches of any one base. Primer extension will occur during the annealing step. Primers are always present in an excess concentration in conventional (symmetric) PCR amplification and, typically, are within the range of 0.1M to 1M. It is generally advisable to use purified oligomers of the highest chemical integrity. Primer Dimers A Primer Dimer (PD) consists of primer molecules that have attached or hybridized to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In the first step of primer dimer formation, two primers anneal at their respective 3 ends. The DNA polymerase will bind and extend the primers. In the third step, a single strand of the product of step II is used as a template to which fresh primers anneal leading to synthesis of more PD product. Primer dimers may be visible after gel electrophoresis of the PCR product. In ethidium bromide stained gels, they are typically seen as 30-50 base-pair (bp) bands or smears of moderate to high intensity. They can be easily distinguished from the band of the target sequence, which is typically longer than 50 bp. One approach to prevent PD formation consists of physical-chemical optimization of the PCR system, i.e., changing the concentration of primers, MgCl2, nucleotides, ionic strength and temperature of the reaction. Reducing PD formation may also result in reduced PCR efficiency. To overcome this limitation, other methods aim to reduce the formation of PDs only. These include primer design, and use of different PCR enzyme systems or reagents. Nuggets Oligonucleotide primers are short fragments of single stranded DNA (17-30 nucleotides in length) that are complementary to DNA sequences that flank the target region of interest. They dictate which region of DNA in the PCR will be amplified. Primer sequences can be obtained by reviewing previously published literature. A confirmation of the sequence can be done by using BLAST (Basic Local Alignment Search Tool). The melting temperature is the midpoint in the observed transition from a double stranded to a single stranded form. A higher annealing temperature ensures that the annealing between the target DNA and the primer is specific. A primer dimer consists of primer molecules that have attached or hybridized to each other because of strings of complementary bases in the primers. Taq polymerase amplifies the primer dimer leading to competition for the PCR products. Several methods are used to reduce primer dimer formation including changing the concentrations of primers, MgCl2, nucleotides, ionic strength and temperature of the reaction. TAQ DNA POLYMERASE The initial PCR reaction used the Klenow fragment of Escherichia coli DNA polymerase. However, this was unstable at high temperatures and it was necessary to add a fresh aliquot of enzyme after every denaturation step. The annealing and extension temperatures had to be kept low and as a result, there was formation of non specific products in abundance. The discovery of the thermostable Taq DNA polymerases ensured that the PCR did not remain a laboratory curiosity. The extension and annealing temperatures could now be kept high and the formation of non specific products was greatly reduced. Taq became famous for its use in the polymerase chain reaction and was called the Molecule of the Year by the journal Science. Why Taq? Taq is the enzyme of choice in PCR because of the following reasons: Taq works best at 75 °C80 °C, allowing the elongation step to occur at temperatures which make non-Watson-Crick base pairing a rare event. It can add upto 1,000 nucleoside triphosphates to a growing DNA strand. Taq has a half-life of 40 minutes at 95 °C and 9 minutes at 97.5 °C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72 °C. Because of all these properties, Taq is the enzyme of choice in the PCR. How does Taq polymerase act? The first requirement is a primer. The primer is annealed to the template strand having free hydroxyl group at its 3 end. During the extension phase, the Taq synthesizes a new DNA strand complementary to the template by adding dNTPs in a 5 to 3 direction condensing the 5 phosphate group of the dNTPs with the 3 hydroxyl group of the end of the extending DNA strand. Since Taq works best between 70 °C- 80 °C, a temperature of 72 °C is usually chosen as the optimum annealing temperature. Where does Taq come from? In Thermus aquaticus, Taq polymerase is expressed at very low levels and commercial production is not economically viable. However, the enzyme can now be produced from different versions of the engineered Taq gene so as to obtain high levels of expression in E coli. What other polymerases are available for use in PCR? Taq is not the only polymerase; other polymerases are available but Taq is the one that is generally used in a PCR. A few other polymerases with their uses are as follows: PFU DNA polymerase -Found in Pyrococcus furiosus, it functions in vivo to replicate the organisms DNA. The main difference between Pfu and alternative enzymes is the Pfus superior thermostability and proofreading properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3 to 5 exonuclease proofreading activity, meaning that it works its way along the DNA from the 3 end to the 5 end and corrects nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. As a result, Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq. However, Pfu is slower and typically requires 1-2 minutes to amplify 1kb of DNA at 72 ° C. Pfu can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity. TFL DNA polymerase Obtained from Thermus flavus, it is useful for the amplification of large segments of DNA. WHAT IS FIDELITY? All DNA polymerases have an intrinsic error rate that is highly dependant on the buffer composition, pH of the buffer, dNTP concentration and the sequence of the template itself. The types of errors that are introduced are frameshift mutations, single base pair substitutions, and spontaneous rearrangements. Therefore, the PCR reaction generates a product that is very similar, but in many cases, not identical to the original sequence. The quantity of dissimilar product obtained is obviously related to the cycle in which the mismatch took place. Under normal circumstances, this does not make any difference; however, these errors may become significant during sequencing when the role of fidelity comes into play. Fidelity is the ability of the polymerases to avoid the incorporation of wrong nucleotides during the reaction. Under normal circumstances, it really does not make a difference if a wrong nucleotide is incorporated because the size of the PCR product remains the same and that is what we have to look for. However, there are some polymerases like Pfu which have a high fidelity. In addition to reading from the 5 to the 3 direction, they can also read from the 3 to the 5 direction and correct the wrong nucleotides wh

Functional Requirements

1.Functional Requirements Functional requirements define the fundamental actions that system must perform.The functional requirements for the system are divided into three main categories, Reservation/Booking, Food, and Management. For further details, refer to the use cases. EXAMPLE 1.1. Reservation/Booking 1.1. The system shall record reservations. 1.2. The system shall record the customer's first name. 1.3. The system shall record the customer's last name. 1.4. The system shall record the number of occupants. 1.5. The system shall record the room number. 1.6. The system shall display the default room rate. 1.6.1. The system shall allow the default room rate to be changed. 1.6.2. The system shall require a comment to be entered, describing the reason for changing the default room rate. 1.7. The system shall record the customer's phone number. 1.8. The system shall display whether or not the room is guaranteed. 1.9. The system shall generate a unique confirmation number for each reservation. 1.10. The system shall automatically cancel non-guaranteed reservations if the customer has not provided their credit card number by 6:00 pm on the check-in date. EXAMPLE 22.Food 2.1. The system shall track all meals purchased in the hotel (restaurant and room service). 2.2. The system shall record payment and payment type for meals. 2.3. The system shall bill the current room if payment is not made at time of service.The system shall accept reservations for the restaurant and room service. EXAMPLE 33. Management 3.1. The system shall display the hotel occupancy for a specified period of time (days; including past, present, and future dates). 3.2. The system shall display projected occupancy for a period of time (days). 3.3. The system shall display room revenue for a specified period of time (days). 3.4. The system shall display food revenue for a specified period of time (days). 3.5. The system shall display an exception report, showing where default room and food prices have been overridden. 3.6. The system shall allow for the addition of information, regarding rooms, rates, menu items, prices, and user profiles. 3.7. The system shall allow for the deletion of information, regarding rooms, rates, menu items, prices, and user profiles. 3.8. The system shall allow for the modification of information, regarding rooms, rates, menu items, prices, and user profiles. 3.9. The system shall allow managers to assign user passwords. 2 Nonfunctional Requirements Functional requirements define the needs in terms of performance, logical database requirements, design constraints, standards compliance, reliability, availability, security, maintainability, and portability. EXAMPLE 1Performance Requirements Performance requirements define acceptable response times for system functionality.The load time for user interface screens shall take no longer than two seconds.The log in information shall be verified within five seconds.Queries shall return results within five seconds.Example Logical Database Requirements The logical database requirements include the retention of the following data elements. This list is not a complete list and is designed as a starting point for developmentBooking/Reservation SystemCustomer first nameCustomer last nameCustomer addressCustomer phone numberNumber of occupantsAssigned roomDefault room rateRate descriptionGuaranteed room (yes/no)Credit card numberConfirmation numberAutomatic cancellation dateExpected check-in dateExpected check-in timeActual check-in dateActual check-in timeExpected check-out dateExpected check-out timeActual check-out dateActual check-out timeCustomer feedbackPayment received (yes/no)Payment typeTotal BillFood ServicesMealMeal typeMeal itemMeal orderMeal payment (Bill to room/Credit/Check/Cash)EXAMPLE 3Design Constraints The Hotel Management System shall be a stand-alone system running in a Windows environment. The system shall be developed using Java and an Access or Oracle database3. Illustrate a timeframe needed to complete each task based on the requirements from question 2.(5 Marks)Answer Estimating time framesTo manage your time well, you should know not only what tasks you need to accomplish, but also when those tasks must be completed and how long they'll take. Making accurate estimates about how long a task will take is one of the keys to effective time management. Many management problems are the result of unrealistic estimates of how long it will take to complete specific tasks.If you estimate time frames accurately, you'll be able to schedule work efficiently and meet deadlines:†¢ schedule work efficiently – Accurate estimates about how long tasks will take to complete make scheduling a lot easier. They ensure that you won't have to keep changing your schedule. If you have a task that you accurately estimate will take six hours, for example, you can allot that time in your schedule and be reasonably confident you won't have to change the schedule. But what if you didn't accurately estimate the time for that task and allotted it only three hours? It would throw your schedule off, and you'd need to rework it.†¢ meet deadlines – If you're accurate in estimating the time it will take to complete tasks, you'll be better able to meet your deadlines. If you're estimates aren't accurate, you may need to ask to change deadlines or disappoint others who are relying on you to complete certain tasks. With accurate time estimates, you'll also be more confident about setting deadlines because you know that the time you assign for completing each of your tasks is realistic.Time estimate equationIt's important to estimate the time frames for your tasks accurately so that you can schedule all your work effectively and meet deadlines. To go about doing this, you first need to know the requirements of each task and your experience with activities – both when they run smoothly and when they don't – to produce three time estimates:†¢ The likely time is the time that the task normally takes you to complete. It helps to consider the time it takes to complete the task without interruption. You should also think about a time frame you would be comfortable with based on your workload, the task, and any external factors that may delay or speed up the completion of the task.†¢ The shortest time is the least amount of time that you have taken to complete the task in the past. It may also refer to the shortest time in which you think you can complete the task if there are no interruptions or distractions.†¢ You can estimate the longest time by considering what may go wrong when performing the task and then adding this extra time to the task's likely duration. This estimate should be based on your experience of this type of activity in the past, as well as on any foreseeable difficulties.You use the three time estimates to calculate the shortest possible time to complete a task based on an average of the likely, shortest, and longest times. Because in most cases a task will take the likely time to complete, this time is given more weight. You need to multiply it by 4, add the shortest time, and then add the longest time. You divide the total by 6 to get the shortest possible time.One important thing to remember is that you must use the same measurements for each type of time. For example, if your likely time is a number of days, the shortest and longest times must also be in days. If your estimates are in different measurements, start by changing them so they are all the same. The time frames equation often produces a shortest possible time that is longer than the shortest time you put into the equation. This is because the equation helps ensure that you're realistic about how long things will take.To manage your time effectively, you have to estimate the time it will take to complete each of your tasks. Doing this ensures you can schedule your work appropriately and meet all your deadlines. To estimate the time frames for your tasks, you can use a simple time frames equation, which uses estimates for the likely, shortest, and longest times to calculate the realistic, shortest possible time that it will take to complete a task.https://library.skillport.com/courseware/Content/cca/pd_11_a02_bs_enus/output/html/sb/sbpd_11_a02_bs_enus002005.html4. Identify and explain five (5) threats to your business that you need to consider for the success of this system.Answer: After assessing the strengths and weaknesses of your business for your business plan, look for external forces, like opportunities and threats, that may have an effect on its destiny. These changes includeThe appearance of new or stronger competitorsThe emergence of unique technologiesShifts in the size or demographic composition of your market areaChanges in the economy that affect customer buying habitsChanges in customer preferences that affect buying habitsChanges that alter the way customers access your businessChanges in politics, policies, and regulationsFads and fashion crazesList the threats and opportunities facing your business, and follow these guidelines:When listing opportunities, consider emerging technologies, availability of new materials, new customer categories, changing customer tastes, market growth, new uses for old products (think about how mobile phones and even eyeglasses now double as cameras and computers), new distribution or location opportunities, positive changes in your competitive environment, and other forces that can affect your success.When listing threats, consider the impact of shrinking markets, altered consumer tastes and pu rchase tendencies, raw material shortages, economic downturns, new regulations, changes that affect access to your business, and competitive threats, including new competing businesses and competitive mergers and alliances. Also think about the impact of expiring patents, labor issues, global issues, and new products that may make your offering outdated or unnecessary.If you're having a tough time getting specific, look back at the strengths and weaknesses, but this time, use it to list strengths and weaknesses of a competitor. You won't know as much about your competitor's capabilities as you know about your own, but you probably know enough to flag areas of strength and weakness. Your competitor's strengths are potential threats to your business, and its weaknesses present potential opportunities.http://www.dummies.com/business/start-a-business/business-plans/how-to-identify-opportunities-and-threats-in-business-planning/5. Write down three (3) elements of risk and two (2) example each that relate to the project.(9 Marks)Answer. All risk management standards agree that the goal of risk management is to enhance the chances of success of the relevant endeavor. However, each of them provides a different definition of risk: ISO31000:2009 calls it â€Å"effect of uncertainty on objectives,† the PMI â€Å"PMBOK Guide† has â€Å"an uncertain event or condition that, if it occurs, has a positive or negative effect on the project's objectives,† and the preferred Risk Doctor definition is â€Å"uncertainty that matters.†Each description is true, but only partly so. This matters because, until we know what we are dealing with, we cannot manage it in the best way possible:If we use the ISO definition, then our first thought will be to focus on the effect;If we follow PMI, then we will start from the potential occurrence;With the Risk Doctor definition, we start from uncertainty.Each of these — the effect, the event and the uncertainty — is a component of risk, but on its own is not a risk.Even taken in pairs they do not provide the full picture:an effect plus an event is an issue;an event plus an uncertainty is a prediction;†¢ an uncertainty plus and effect is a concern.It is only when you put all three together that you can see what a risk is made of, and use this information to decide on what, if anything, to do about it. Of course, this then requires a longer definition, but the goal enhancing the chances of success is worth the effort.But what is â€Å"success†? It is more than simply â€Å"meeting objectives;† it must also include the condition of â€Å"complying with project constraints† in order for the final result to remain within scope. Given this clarification, a more complete definition is: â€Å"Risk consists of three parts: an uncertain situation, the likelihood of occurrence of the situation, and the effect (positive or negative)that the occurrence would have on project success.†The three-part definition helps with three important stages of the risk management process: In 1.risk identification, it supports the structured description of a risk (â€Å"risk metalanguage†) in the form: â€Å"Because of , may occur, leading to In 2.risk evaluation, knowledge of potential causes allows you to evaluate the likelihood; identification of effects provides a basis for quantifying the impact.In 3 risk response planning, the different parts of the definition suggest different response approaches:for threat avoidance, understanding the situation may allow you to stop it happening or protect against its results;understanding the situation can also be used to help us exploit opportunities;in risk transfer or sharing, we seek a partner better equipped to address the effect;for threat reduction or opportunity enhancement, we focus on the effect and/or the likelihood;in risk acceptance, any contingency plan has to address the effect.Including these three components when you describe risks (the uncertainty, the event and the effect) will help everyone involved in risk management to take account of these three important aspects of risk, and act on them to enhance the chances of success.EXAMPLETwo examples of Managing risk in hote

Speaking Spanish Has Always Been A Struggle

She played a huge role in teaching me how to speak Spanish. The only language she would speak to me while growing up was Spanish. As I grew up I never realized the method that was being used which was direct method until now. She was teaching me on how to communicate with others while speaking another language. My grandmother only spoke to me in Spanish though I had a rough time understanding and trying to speak it back to her, she was determined to teach me.The role of the teacher who in this case is my grandmother is to teacher me on how to become a communicator. Not merely to learn vocabulary or grammar but to know whom, when and what to say or how to ay certain things. The role of my grandmother was to acquire a learning system which was not focused too much on how to write or learn vocabulary but to learn the proper way to use Spanish first. The students role is to accept the corrections as well as absorbing and putting my Spanish speaking skills to test.Though till this day I a m not that great at speaking my second language, know the basic fundamentals of this language, my language in this case would be â€Å"Spangling†. This is where I incorporate both Spanish and English while speaking to Spanish speakers, which my grandmother isn't too fond of. One characteristic that is good for teaching Spanish would be proficiency, in order to teach a foreign language you must know the language yourself. One must learn how to speak it, read it as well as write it. It was easy for my grandmother to teach me because she was well educated in that language.She was able to exhibit a high level of accuracy in Spanish because of her background with the language. As for the learning process I was able to surround myself with other family members to communicate and improve in my foreign language. As well as watching my grandmothers intense Spanish wows which educated me more and taught me on how to express myself. The nature interaction between my grandmother and I wa sn't too bad, though I struggled in the beginning as time went by I was able to learn it eventually, not as good as I had wished though.It was harder for my because growing up because had a New Yorker accent so for some words had a hard time saying in Spanish, and for that my grandmother made fun of me but thought it was adorable. Plenty of corrections were made during my process of learning Spanish which better my knowledge and understanding in this language. While learning Spanish it was pretty difficult and frustrating especially since I had an accent which was harder for me to comprehend in also my sisters made fun of me because of it Especially when it came to rolling the Or's that was the most difficult part of Spanish was being able to roll letters.As well as learning on how to speak very fast in my language, normally all Spanish speakers have a tendency to speak very fast, still haven't gotten the hang of that but I'm learning. When speaking Spanish I have made plenty of mis takes but I was able to remind myself that it's okay to make mistakes, but to ask ND seek help from people who are great in speaking Spanish. Speaking Spanish in my household was and still is a must, the only way to communicate to my grandmother was through speaking Spanish.Especially since it is my second language it is important that I know how to speak it. Am also able to expand my horizons and know more and how my language works. It's not just about learning a new language and how to speak it but you learn about its culture as well. It's important so we can empathic with people in our country who struggle to learn English, so therefore learning more engages and about different cultures will help us develop more knowledge of other languages. The area of language that is emphasized are vocabulary and grammatical structure.Vocabulary is very important for every language especially so you're able to use it in the correct form. I know for myself whenever use the incorrect word while speaking to my grandmother she looks at me with a confused face and waits for me to correct myself. Vocabulary is an important factor because you want your sentences understandable while using the right terms. As for grammatical, its important to govern the compositions of harass, clauses and words so you can acquire a better sense of how to use these while speaking to an individual.As a child my grandmother would have me repeat every word after her in Spanish and once we did that, she told me what each word meant. Of course we started off with the easy terms first such as animals, fruits and parts on our bodies. As we did those every day as a child my vocabulary increased and little by little began to understand the basics, once we did that we moved onto the harder stuff. Learning different terms helped me improve in this engage and gave me a better sense on how to use them and when to use them. As only able to speak Spanish whenever I would see my grandmothers, or went to visit pe ople in my country. My native language which is English is Of course my strongest skill but speaking Spanish has given me the opportunity to explore more in my foreign language and not rely so much on my native language. The role of my native language should not be used when learning another language, should be able to get out of the habit of not depending so much on it and being able to be open to learning different languages especially my own language.

Los 6 puntos bsicos de las visas J-1 de intercambio

Cada mes hay una media de 170,000 extranjeros disfrutando de una visa J-1 de intercambio en los Estados Unidos. Si se tiene una, es muy importante conocer las reglas fundamentales. En este artà ­culo se informa sobre los programas amparados bajo el nombre J-1, explicacià ³n de fecha mà ¡xima para permanecer legalmente en el paà ­s y periodo de gracia, quà © es la regla de la obligacià ³n de dos aà ±os fuera, cà ³mo saber si aplica al caso de cada uno y, en el caso de los mà ©dicos, cuà ¡les son los caminos para pedir una waiver, tambià ©n conocido como perdà ³n o permiso.   Para qu es la visa J-1 de intercambio Para facilitar que personas extranjeras ingreses a Estados Unidos a participar en diversos programas de intercambio educacional o cultural. Los Programas de la visa J-1 Son muy diversos entre sà ­ y con condiciones comunes pero tambià ©n à ºnicas a cada programa. Au-pair o nià ±eraEstudiante universitario, dentro de un programa encaminado a completar los estudios universitarios que realiza en su paà ­s de origenStaff  de campamento de verano Visitante invitado por el gobierno federal, uno de los estados o municipales o una de sus agenciasPrà ¡cticas para profesionales en Estados UnidosProgramas de entrenamiento para profesionales, como este para ONGs en EEUU. Mà ©dicos para realizar estudios o prà ¡cticasProfesores universitarios o investigadores a medio plazo (Visas H-1B y en algunos casos la visa O son tambià ©n una opcià ³n).Profesores universitarios o investigadores por un periodo corto de tiempo, por ejemplo, una conferencia, una demostracià ³n, etc.Estudiantes de secundaria para cursar sus estudios en una institucià ³n pà ºblica o privada. Para estos estudiantes, hay otras opciones de visa.Especialistas en un à ¡rea concreta de conocimientoTrabajar y viajar en verano para universitariosMaestros (los maestros tambià ©n pued en obtener una visa H-1B) Cul es el primer paso para obtener una visa J-1 Tener un patrocinador (sponsor). Sà ³lo pueden patrocinar una J-1 una institucià ³n pà ºblica o privada que haya sido designada especà ­ficamente por el gobierno americano. Por ejemplo, aquà ­ està ¡ la de patrocinadores de maestros y aquà ­ una lista completa para otros programas. Si el sponsor decide patrocinar enviarà ¡ al patrocinado extranjero un documento que se conoce como DS-2019. Si este documento no se puede pedir la visa. Solicitud de la visa A partir de ahà ­ ya se puede solicitar la J-1 completando en là ­nea el documento DS-160 pagando la tarifa correspondienteaportando los documentos especà ­ficos que se piden para cada programa y segà ºn el lugar desde donde se aplica (seguir las instrucciones del DS-160)asimismo, aportar las fotos y huellas digitales siguiendo las pautas del consulado desde donde se aplica.  acudir al consulado con toda la documentacià ³n y los resguardos de haber pagado haber aplicado el dà ­a y la hora seà ±alado. Seguir en este punto las instrucciones concretas para cada oficina consular Se puede llevar a Estados Unidos a los familiares mientras se desarrolla el programa de intercambio? En la mayorà ­a de los casos es posible solicitar una visa J-2 para los esposos/as   y para los dependientes (hijos solteros menores de 21 aà ±os). Sin embargo, algunos programas no contemplan esta posibilidad son: au pairmonitor de campamento de veranoestudiante de secundariay programa de trabajar y viajar en verano El procedimiento para obtener la visa J-2 es igual que al de la J-1. Primero hay que obtener el OK del patrocinador y este debe dar el DS-2019 para cada dependiente. Y sà ³lo despuà ©s se solicita la visa per se. En la mayorà ­a de los casos los cà ³nyuges que son titulares de una J-2 pueden trabajar en Estados Unidos, para ello deben solicitar un permiso de trabajo despuà ©s de ingresar al paà ­s.  En cuanto a la validez de sus visas, dejarà ¡n de serlo cuando la J-1 de la que dependen no sea và ¡lida Obligacin de salir del pas al completar el programa J-1 y no regresar por dos aos Algunos programas està ¡n sujetos a este mandato cuando la participacià ³n en el programa recibe fondos del gobierno del paà ­s del patrocinado o de los Estados Unidoscuando se trata de mà ©dicoscuando se posee unos conocimientos o habilidades calificados como necesarios por parte del paà ­s al que pertenece la persona que disfruta de la visa J-1. En estos casos ha que salir de Estados Unidos por dos aà ±os, aunque se puede viajar como turista. Sin embargo no se puede obtener una visa de trabajo tipo L, H o ni una tarjeta de residencia ya que no es posible obtener ni una visa de inmigrante ni un ajuste de estatus. Tampoco es posible sacar la visa K-1 para prometidos de ciudadanos americanos. Los à ºnicos transfer de visa posibles son por una G, para organizaciones internacionales, una A, para diplomà ¡ticos o la visa U para ciertos casos de và ­ctimas de violencia. Es cierto que se puede solicitar una waiver para que esta prohibicià ³n no aplique, pero no es fà ¡cil obtenerla, por lo que es altamente recomendable asesorarse con un abogado migratorio. Cmo saber si se est sujeto a la regla de los dos aos Se puede verificar en la esquina inferior izquierda del documento DS-2019, aunque es posible que està © en blanco.   En todo caso, la visa J-1 sà ­ que menciona explà ­citamente si se està ¡ sujeto a esta regla de tener que salir de los Estados Unidos por dos aà ±os y es que eso aplica a las personas cuyas visas tengan las palabras: Visa Bearer is suject to the 212 (E) Two year residency rule does apply.   Waivers para mdicos Los mà ©dicos con una J-1, en teorà ­a, deberà ­an cumplir con la regla de los dos aà ±os de residencia fuera de Estados Unidos. Pero como es una profesià ³n en gran demanda, principalmente en ciertas zonas del paà ­s, se admiten varios caminos para pedir una waiver: Programa Conrad-30, que permite solicitar a cada uno de los 50 estados un total de 30 waivers.Programa HPSA, gestionado por el Departamento de Salud y Servicios Humanos. Programa ARC, gestionado por la Comisià ³n Regional de la Apalachiay el Programa Delta, presente en lo estados de Alabama, Arkansas, Illionois, Kentucky, Louisiana, Mississippi, Missouri y Tennessee. Entradas y salidas con la visa J-1 Para evitar tener la visa cancelada o que impidan el ingreso a los Estados Unidos es muy importante conocer cuà ¡ndo se puede ingresar al paà ­s, cuà ¡nto se debe salir y cuà ¡l es el periodo de gracia. Quedarse mà ¡s tiempo del permitido tiene como consecuencia la cancelacià ³n de la visa y, a partir de los 180 dà ­as de estancia ilegal la situacià ³n se puede complicar por aplicacià ³n del castigo de los 3 y de los 10 aà ±os.   Informacin adicional Si la visa J-1 no es la adecuada para un posible visitante a los Estados Unidos, un buen lugar por donde comenzar a informarse es conocer todas las visas no inmigrante con las que se puede ingresar al paà ­s. En algunos casos, los titulares de una visa J-1 pueden traer a sus familiares con una visa J-2. En el caso de nià ±os en primaria o secundaria es muy recomendable familiarizarse con el sistema educativo de los Estados Unidos antes de llegar al paà ­s.   Este artà ­culo no es informacià ³n legal para ningà ºn caso en concreto. Es sà ³lo informacià ³n general.