SNPWizard User Guide

Transcription

1 SNPWizard User Guide About SNPWizard There are many situations in which one wishes to amplify a small segment of DNA where otherwise identical strands may differ. Such segments may consist of a single base whose variation is the sole distinguishing characterstic of two functionally distinct sequences, a single nucleotide polymorphism, hence SNP. For the purpose of detecting, identifying, and quantifying these variants, it is useful to design and optimize primers for RT-PCR which bound the region of variation as closely as possible yet do not misprime with each other or anywhere in the amplicon or nearby on the template DNA. SNPWizard.vi is a Labview based front-end user application developed by Carl Wittwer and Bob Palais that accomplishes this task. The user enters a sequence by pasting or selection from a.aln (Clustal format) file and provides one or more altered sequences by interactively editing bases in the initial sequence. The user also enters specific PCR conditions to be used (concentrations of primers, target DNA, buffers, etc.) SNPWizard then identifies the smallest segment containing all of the changes and iteratively builds candidate forward and reverse primers outward from these locations to minimize the number of consensus bases versus bases which vary between amplicons. Candidate forward primers identical to the consensus sequences before the mutation region and reverse primers complentary to the consensus sequences beyond the mutation region are constructed to have the minimal length satisfying the primer annealing Tm (default 60 Celsius) designated by the user. SNPWizard then eliminates all candidate primers which individually would tend to form homodimers (self-anneal) or misprime (anneal to locations in DNA which might be present other than the intended location.) It pairs forward and reverse primers from the remaining candidates and eliminates those pairs which would tend to form heterodimers (cross-anneal). The dimerization condition is surprising, but based on considerable experience, we eliminate any primer (or pair) with two 3 end bases complementary to itself (or to each other), and at least one of the two is a G or a C. This criterion sets an annealing temperature under the specified PCR conditions, and if any possible configuration (shift) of the 3 ends oligo pair in question with respect to each other leads to an annealing temperature below this value, it is eliminated for potential dimerization. The reason for this is that if such an annealing were to take place, rare as it may be at the high temperatures of the RT-PCR cycle, the dimer formed would immediately extend to form strands containing longer segments complementary to both primers and which would amplify and create ever more misprimes at ever higher Tms. Primer pairs which can misprime (i.e., anneal to sections of the amplicon or other specified DNA other than the intended DNA locus above a user defined threshhold-default 10 Celsius- below intended annealing temperature under prescribed conditions present during PCR) are also eliminated. If this type of misprime occurs, alternate amplicons could corrupt the desired product, or at the very least, primers will be sidelined and unable to amplify the intended amplicons exponentially. If dimers or misprimes are found, additional candidate forward and reverse primers are built whose 3 ends are one base further from the polymorphic segment than at the 1

2 previous stage. All possible pairs of forward and reverse primers that have been built thusfar are again tested for misprimes. When a user specified number of pairs which pass the dimer and misprime tests are found, the search terminates. If not enough primers can be found, it terminates an alerts the user that fewer or no pairs were found. The user may then view and output the list of primer pairs found, along with their locations, primer Tms, amplicon Tms with respect to the various sequences entered, the minimum Tm separation of these amplicons, and data regarding misprimes which have been found. After SNPWizard is run for a particular set of sequences with a particular set of misprime parameters, the user has the option of resetting the misprime parameters and finding a new choice of primers for the same sequences subject to the new parameters. All of the information necessary to reproduce the original and altered sequences and misprime parameters for each run, as well as the output data for any of the primer pairs discovered by SNPWizard (primer sequences, locations, and Tms, amplicon Tms with each sequence and minimum separation) may be output to a spreadsheet for subsequent use. All Tms in SNPWizard are based upon an improved implementation of the nearestneighbor thermodynamic models described in SantaLucia Jr., et. al. [1-8], adapted to more common and realistic PCR conditions. In these papers, Tm represents the predicted temperature at which half of the DNA is coiled and half uncoiled. In this and other aspects, SNPWizard is a close relative of MultiPrimer [9], a program that designs and optimizes primers to detect, identify, and quantify distinguishing amplicons within sets of multiply aligned sequences having considerable consensus while still containing multiple sites of polymorphism. The differences between SNPWizard and MultiPrimer can be described as follows: SNPWizard looks for primers bounding one locus of polymorphism on otherwise identical sequences while MultiPrimer compares the effect of primers bounding polymorphic regions throughout several similar sequences. SNPWizard develops several candidate primer pairs surrounding the single polymorphic region while MultiPrimer develops one forward and one reverse primer for every consensus segment among sequences. SNPWizard builds the shortest acceptable primers from each starting point, and all sequences agree where these primers bind, while MultiPrimer sometimes restricts or uses consensus segments and other times finds the most stable primer within a set temperature range, with respect to the Tms of similar yet varying sequences to which they anneal. MultiPrimer also restricts primers based upon user input sequences they should not amplify, which SNPWizard does not, and a such MultiPrimer has the ability to build primers based upon favorable locations for mismatches with these sequences. Since SNPWizard does not deal with multiply aligned sequences, it has no need to treat alignment markers (e.g. hyphens) in its sequences. To run SNPWizard, simply set the switch for the input mode (paste the basic sequence or read it from an.aln file) and click the Enter Sequences button. Pasted sequences will have all but the letters a,c,g,t,a,c,g, or T removed, and lower case letters converted to upper case automatically. Next, a window will appear allowing the user to interactively 2

3 add sequences to alter, and changes their bases at selected sites. The changes are visible as they are made, and the user may delete sequences if they wish, and extreme base positions will be correctly recomputed. The program will prevent the user from attempting to change bases outside the range of the sequence, or from proceeding to the primer search phase if no sequences have been created or bases altered. When the user indicates that they are finished creating altered sequences, they are returned to the main window where they may alter the default misprime search parameters before clicking Find Primers to have the program perform its search. Results may be written to a window that may be saved to a.xls spreadsheet file at any time. The user can choose to modify the misprime search parameters for the same sequences and run a new search. Previous results may be erased from the window or the new misprime parameters and results appended to previous ones. Primer-sequence misprimes are reported with their location and Tm, while primerprimer misprimes are reported with which of the possible pairs misprime: FFr (forward with forward reversed), FRr (forward with reverse reversed), RRr (reverse with reverse reversed) and the size of the smallest overlap that generates the misprime. 3

4 How melting temperatures are modeled The melting temperature, T M, at which half of the strands are in the double-helical state and half in random-coil state is predicted using the nearest-neighbor thermodynamic model and experimental parameters reported in SantaLucia Jr. et. al. [1-8] where a phase transition is predicted by 0 = dg = dh TdS so T M = dh ds = ( H tetrads + ends )/( S tetrads + ends + R lnc T +.368N ln[na + ]) where dh and ds are the contributions to free energy in kcal/mol and to entropy in cal/kelvin*mol from the internal configuration of the oligonucleotide (interior tetrads and end pairs), R is the gas constant (1.987 cal/kelvin*mol), C T is the difference of the larger and half the smaller oligonucleotide strand concentrations (target and probe), N is one half of the total number of phosphates in the duplex (e.g. for an 8-bp duplex without terminal phosphates, N=7). The module TM Calculator.vi estimates the melting temperature by calling Thermo- DynamicOligos.vi which in turn refers to lookup tables of contributions to H and S between double-helical and random-coil states. dhdsinit.vi contains the values for A-T or G-C terminal ends. ThermodynamicPairs.vi contains the data for internal tetrads: 16 configurations with two (neighboring) matched pairs ([1]), 64 with one matched pair and one unmatched pair (16 for each unmatched pair, G-T ([2]), G-A ([3]), A-C ([4]), C-T ([5]), and 32 with AA,CC,GG,TT mismatches ([7]) ), and 32 tetrad configurations with one match adjacent to a dangling end ([8]). 4

5 References 1. SantaLucia, Jr., John, Allawi, Hatim T, and Seneviratne, P. Ananda, Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability. Biochemistry 1996, 35, Allawi, Hatim, and SantaLucia, Jr., John Thermodynamics and NMR of Internal G-T mismatches in DNA. Biochemistry, 1997, 36, Allawi, Hatim, and SantaLucia, Jr., John Nearest Neighbor Thermodynamic Parameters for Internal G-A Mismatches in DNA. Biochemistry, 1998, 37, Allawi, Hatim, and SantaLucia, Jr., John Nearest-Neighbor Thermodynamics of Internal A-C Mismatches in DNA: Sequence Dependence and ph Effects. Biochemistry, 1998, 37, Allawi, Hatim, and SantaLucia, Jr., John Thermodynamics of internal C-T mismatches in DNA. Nucleic Acids Research, 1998, Vol. 26, No. 11, SantaLucia, Jr., John A unified view of polymer, dumbell, and oligonucleotide DNA nearest-neighbor thermodynamics, Proc. Natl. Acad. Sci. USA, Feb. 1998, Vol 95. pp Peyret, Nicolas, Seneviratne, P. Ananda, Allawi, Hatim T, and SantaLucia, Jr., John, Nearest-Neighbor Thermodynamics and NMR of DNA Sequences with Internal AA,CC,GG,TT mismatches. Biochemistry 1999, 38, Bommarito, Salvatore, Peyret, Nicholas, and SantaLucia, Jr., John Thermodynamic parameters for DNA sequences with dangling ends. Nucleic Acids Research, 2000, Vol. 28, No. 9, Wittwer, Odell, Palais, Sanders, MultiPrimer: A primer design and optimization tool for multiply aligned sequences. 5