development of Serial Analysis of Gene Expression (SAGE), a powerful quantitative technique for profiling gene expression.
In the clinical arena, there are three main diagnostic applications of PCR: detection of pathogens, screening specific genes for unknown muta-tions, and genotyping. For instance, PCR permits mutation detection and early identification of deadly infective agents like SARS, small pox, HIV, and influenza. PCR can be applied for tumor cell detection as well, mixed chimerism after bone marrow transplantation, and noninvasive prenatal screening of fetal DNAin the maternal circulation. Single-strand confor-mational polymorphism (SSCP) is one of the most widely used methods for detecting single base pair mutations. SSCP is based on the sequence-dependent intramolecular folding that is responsible for the differential migration of two single-stranded DNA molecules of identical length but dissimilar sequence on nondenaturing acrylamide gel. Finally, efficient extraction of RNA and DNA from formaldehyde fixed tissues was recently achieved and, importantly, it was shown that it is possible to perform PCR on these often-damaged short DNAs. This accomplishment gives researchers the exciting opportunity of utilizing PCR to obtain informa-tion from archival materials collected many years ago, thus contributing to the understanding of common complex, chronic diseases.
Applications of real-time PCR include, among others, gene expression quantification and detection, validation of gene expression data obtained by microarray analysis, measurement of DNA copy number, detection and quantification of viral particles and potentially lethal microorgan-isms, allelic discrimination, and SNP detection.
4. PCR Primer Design
Successful PCR relies on the utilization of suitable primers. Human-designed PCR primers often fail due to low amplification efficiency, non-specific amplification, and primer-dimers formation. Several programs are available that help design the most appropriate set of primers for a given application; many of these tools are freely available online. Several companies that synthesize custom oligos usually offer either in-house, commercial, or academic sites for primer design. With these computa-tional tools, primer pairs are computed from user-selected target regions and then screened against a series of parameters to maximize priming efficiency for trouble-free PCR. The following attributes are included in primer design: target sequence, amplicon length, cross homology with related genes and pseudogenes (if present), amplicon location (distance
from 3' end), primer Tm (melting temperature), primer length, primer del-taG 3' end, intron spanning, GC content, and primer hairpin structure. For qRT-PCR, added parameters include perfect probe/template anneal-ing Tm, probe 5' and 3' extensions, probe meltanneal-ing point, probe length, and probe GC content. Getting these characteristics right is critical to achiev-ing sensitive, specific, and reproducible PCR results.
In the following text is a list of some online sites for the design PCR primers (see also Table 2.3). Several sites include the development of repositories for primer sets, reaction conditions, and the primers them-selves that would benefit investigators interested in contributing to and taking advantage of the information available:
Primer3 designs PCR primers from DNA sequences according to thermodynamic, primer size, and product size characteristics (Rozen and Skaletsky, 2000). Primer3 software can check existing primers and design hybridization probes as well. This can be use-ful, for example, for spotted arrays for mRNA expression profiling. Primer3 was developed at the Whitehead Institute and the Howard Hughes Medical Institute. The Primer3 Web site is also funded by the National Institutes of Health.
AutoPrime designs primers for real-time PCR for eukaryotic gene expression. Primer pairs are selected in such a way that at least one of them matches an exon-exon border sequence that is present in the cDNA but not in the genomic sequence. Alternatively, the pair is designed by placing each primer in a different exon so that a genomic product would include a long intronic sequence unlikely to be ampli-fied under the running PCR conditions.
RTPrimerDB is a public database for primer and probe sequences used in real-time PCR that employs popular chemistries such as SYBR Green I, Taqman, hybridization probes, and molecular •
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TABLE 2.3 Web Sites of Some Programs That Perform PCR Primer Design
Site Address Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi AutoPrime http://www.autoprime.de/AutoPrimeWeb RTPrimerDB http://medgen.ugent.be/rtprimerdb/index.php PrimerBank http://pga.mgh.harvard.edu/primerbank/index.html QPPD http://web.ncifcrf.gov/rtp/gel/primerdb/
beacons. This site encourages users to submit their validated primer and probe sequences, so that other users can benefit from their expe-rience. The goals of this site are twofold: to prevent time-consuming primer design and experimental optimization (Pattyn et al., 2006), and to introduce a certain level of uniformity and standardization among different laboratories.
PrimerBank is another public resource for PCR primers for gene expression detection or quantification (real-time PCR). It contains about 180,000 primers covering most known human and mouse genes. The company claims that primer design algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency.
QPPD (Quantitative PCR Primer Database) provides information about primers and probes gathered from published articles cited in PubMed. Primers are used to quantify human and mouse mRNA. Part II Step-By-Step Tutorial
The following is a guide to designing primers using the Primer3 program.
1. Sequence Input and Parameters Selection
The first step is to cut and paste the sequence in the input window (Fig-ure 2.7). Note that the entire window has been divided into two fig(Fig-ures, (Figure 2.7 and Figure 2.8). Sequences should be devoid of cloning arti-facts or chimeric sequences and should not contain repetitive elements. Low-quality bases should be changed to N’s or be made part of “Excluded Regions.” “Sequence Id” is an assigned identifier that is reproduced in the output to enable you to identify the chosen primers. For standard PCR, only the “left” and “right” primer options should be selected. In the “Excluded Regions” box, primers will not overlap any region specified in this tag. Enter the value as a space-separated list of start,length pairs, where start is the index of the first base of the excluded region, and length is its length. This feature is useful for excluding regions of low sequence quality or rich in repetitive elements such as ALUs.
Figure 2.8 shows commonly used settings for primer design. “Product Size Range” displays a list of target size ranges (100 bp in the example shown). Primer3 attempts to pick primers starting with the first range, then goes to the next range and tries again. The program continues in this way until the last range is screened or until it has picked all necessary
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FIGURE 2.7 Primer design using Primer3 program. The sequence to be used for primer selection is entered in the open box. (For details of sequence properties, see text). Primer design for standard PCR is shown. If real-time PCR is to be per-formed using Taqman, the hybridization probe can be designed by also selecting the middle box.
FIGURE 2.8 Primer design using Primer3 program (continuation). Commonly used settings for primer design are shown (for details, see text).
primers. Selecting this option is less computationally demanding than the “Product Size” option. For “Product Size,” Primer3 will not generate primers with products shorter than Min (minimum) or longer than Max (maximum) length entered, and will try to pick primers producing ampli-cons close to the optimum length. “Number To Return” is the maximum number of primer pairs displayed in the result document (in this case, 5). The program sorts the primer pairs from best to the poorest “quality.” Choosing a large value in this setting will increase the running time. “Max 3' Stability” is the maximum stability for the five 3' bases of either primer measured as a value of the maximum deltaG for duplex disruption, which is calculated by the nearest-neighbor method. Bigger numbers mean more stable 3' ends. “Max Mispriming” (default is 12) is the maximum allowed weighted similarity with any sequence in Mispriming Library. “Pair Max Mispriming” (default value is 24) is the maximum allowed sum of simi-larities of a primer pair with any sequence in Mispriming Library.
For the “General Primers Picking Conditions” section, enter values in “Primer Size” for minimum, optimum, and maximum primer lengths. Primer3 will attempt to pick primers close to Opt and not shorter than Min or longer than Max (which cannot be larger than 36 bases). The “Primer Tm” option sets the minimum and maximum melting
tempera-tures (in Celsius) for a primer. Primer3 will try to pick primers with melt-ing temperatures (“Primer Tm”) close to Opt and not smaller than Min or
larger than Max. “Maximum Tm Difference” is the maximum acceptable
difference between the “Primer Tm” values. Primer3 uses the oligo
melt-ing temperature formula given by Rychlik et al. (1990) and Breslauer et al. (1986). The “Product Tm” is the minimum, optimum, and maximum
melting temperature of the amplicon. Product Tm is calculated using the
formula from Bolton and McCarthy (1962), which considers the sodium concentration, %GC content, and the sequence length in the calculations.
Primer3 uses penalty components to pick the best primers by taking into consideration values less or greater than those specified as optimum. The score indicates deviation from the specified optimal design param-eters; a lower penalty score indicates a better primer pair. Penalty weights are governed by various parameters, including the Tm difference, primer–
primer complementarity, primer–primer 3' complementarity, and primer pair mispriming similarity. Position Penalty Weight determines the over-all weight of the position penalty in calculating the penalty for a primer. Deviations from the optimum primer size and Tm have a large influence on
primer to anneal to itself or to each other. The scoring system gives 1.00 for complementary bases, −0.25 for a match of any base with an N, −1.00 for a mismatch, and −2.00 for a gap. Only single base pair gaps are allowed. A score of 0.00 indicates that there is no reasonable local alignment between two oligos. “Max 3' Complementarity” tests for complementarity between left and right primers. A score of 0.00 indicates that there is no reasonable 3'-anchored global alignment between two oligos. The score of a local alignment will always be at least as great as the score of a global alignment. “Max Poly-X” refers to the length of mononucleotide repeat.
In “CG Clamp” box, the number of consecutive G’s and C’s at the 3' end of both the left and right primer are specified. When “Liberal base” is checked, Primer3 accepts IUB/IUPAC codes for ambiguous bases (see Table 2.1).
2. Results
After clicking the “Pick Primers” button, a window with a text document (Primer3 output) appears (Figure 2.9 and Figure 2.10).
The window displays a table with the first (i.e., best) left and right primer pair sequences shown on the right (always in 5' to 3') along with their start positions, lengths, melting temperatures, and percentage of G or C bases. Their self- and 3' self-complementarity scores are also displayed. Below the table, the predicted product size is shown as well as the DNA sequence and the positions where the left and right primers indicated by arrowheads map (in the figure, the forward left primer is boxed). Other programs pro-vide extinction coefficient, molecular weight, µg/OD, nmol/OD, predicted secondary structures (hairpins), and potential duplexes when the oligo can anneal to any target sequence.
At the bottom of the window (Figure 2.10), similar information is pro-vided for the remaining four additional primer pairs (note that “Number To Return” was set at 5 [see Figure 2.8]). Some statistics are given regard-ing the number of considered and unacceptable primers.
Some programs offer the possibility of sending your input sequence to NCBI Blast search for short, nearly exact matches. When working with software with multiplex capability, it may be necessary to try several values for melting temperature and %GC content before finding a mul-tiplex primer set for your sequences. The time spent designing the prim-ers should be worthwhile as it will reduce the time in the multiplex PCR optimization step.
Part III Sample Data
The sequence used in the previous demo procedure is shown in the fol-lowing text. It is the mouse SM22alpha gene flanking sequence, accession number L41161.1, GI:793751, for Mus musculus SM22 alpha gene (Solway et al., 1995). Position 1 in exon 1 at nucleotide 1341 is shown underlined in boldface.
FIGURE 2.9 Results displayed using Primer3 program. The output window lists, among others, the primer pair sequences and their properties (start positions, lengths, melting temperatures, and percentage of G or C bases), as well as the predicted amplification product size and their position within the sequence.
1 gaattcagga cgtaatcagt ggctggaaag caagagctct agaggagctc cagcttatta 61 tgacccttcc ttcagatgcc acaaggaggt gctggagttc tatgcaccaa tagcttaaac 121 cagccaggct ggctgtagtg gattgagcgt ctgaggctgc acctctctgg cctgcagcca 181 gttctgggtg agactgaccc tgcctgaggg ttctctcctt ccctctctct actcctttc 241 ccctctccct ctccctctct ctgtttcctg aggtttccag gattggggat gggactcaga 301 gacaccacta aagccttacc ttttaagaag ttgcattcag tgagtgtgtg agacatagca 361 cagatagggg cagaggagag ctggttctgt ctccactgtg tttggtcttg ggtactgaac 421 tcagaccatc aggtgtgata gcagttgtct ttaaccctaa ccctgagcct gtctcacctg 481 tcccttccca agaccactga agctaggtgc aagataagtg gggacccttt ctgaggtggt 541 aggatctttc acgataagga ctattttgaa gggagggagg gtgacactgt cctagtcctc FIGURE 2.10 Results displayed using Primer3 program (continuation). Addi-tional primer pairs are displayed with some of their properties.
601 ttaccctagt gtctccagcc ttgccaggcc ttaaacatcc gcccattgtc accgctctag 661 aaggggccag ggttgacttg ctgctaaaca aggcactccc tagagaagca cccgctagaa 721 gcataccata cctgtgggca ggatgaccca tgttctgcca cgcacttggt agccttggaa 781 aggccacttt gaacctcaat tttctcaact gttaaatggg gtggtaactg ctatctcata 841 ataaagggga acgtgaaagg aaggcgtttg catagtgcct ggttgtgcag ccaggctgca 901 gtcaagacta gttcccacca actcgatttt aaagccttgc aagaaggtgg cttgtttgtc 961 ccttgcaggt tcctttgtcg ggccaaactc tagaatgcct ccccctttct ttctcattga 1021 agagcagacc caagtccggg taacaaggaa gggtttcagg gtcctgccca taaaaggttt 1081 ttcccggccg ccctcagcac cgccccgccc cgacccccgc agcatctcca aagcatgcag 1141 agaatgtctc cggctgcccc cgacagactg ctccaacttg gtgtctttcc ccaaatatgg 1201 agcctgtgtg gagtgagtgg ggcggcccgg ggtggtgagc caagcagact tccatgggca 1261 gggaggggcg ccagcggacg gcagaggggt gacatcactg cctaggcggc ctttaaaccc 1321 ctcacccagc cggcgcccca gcccgtctgc cccagcccag acaccgaagc tactctcctt 1381 ccagtccaca aacgaccaag ccttgtaagt gcaagtcat
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