CODEHOP使用说明

CODEHOP : COnsensus-DEgenerate HybridOligonucleotide PrimersCODEHOP helpOn this page:Other pages: •General information • Getting started •Obtaining input alignments • CODEHOP program algorithm • Terms and parameters • CODEHOP manuscript • Strictness parameter details • Basic tips• PublicationGeneral informationThe CODEHOP program designs PCR (Polymerase Chain Reaction) primers from protein multiple-sequence alignments. The program is intended for cases where the protein sequences are distant from each other and degenerate primers are needed.The multiple-sequence alignments should be of amino acid sequences of the proteins and be in the Blocks Database format Proper alignments can be obtained by different methods .The result of the CODEHOP program are suggested degenerate sequences of DNA primers that you can use for PCR. You have to choose appropriate primer pairs, get them synthesized and perform the PCR.A CODEHOP primer is degenerate at the 3' core region, with a length of 11-12 bp across four codons of highly conserved amino acids, and is non-degenerate at the 5' consensus clamp region, with a length which depends on its desired annealing temperature, typically between 20 and 30bp:5' 3'--------------------===========non-degenerate degenerateconsensus clamp core#bases from temp 11-12 basesThe hybrid structure (5' consensus and 3' degenerate) of CODEHOP primers allow the PCR amplification to be specific during the early cycles from the original source DNA and selective during the late cycles from the PCR synthesized products:Schematic comparison of standard degenerate PCR (left) with the CODEHOP (right), illustrating regions of mismatch in primer-to-template annealing during early PCR cycles and in primer-to-product annealing during subsequent cycles. Vertical lines indicate nucleotide matches between primer (arrow) and template or synthesized product. The overall degeneracy is the product of degeneracies at each nucleotide position, so that the fraction of precisely hybridizing primers is 1/degeneracy.Obtaining input alignmentsInput alignments for the CODEHOP program must be in the Blocks Database format. Block multiple-sequence alignments are ungapped and usually local alignments. Local alignments cover only parts of the protein sequences. The regions between the blocks are the ones with no sequence similarity or where gaps must be inserted to align the sequences.You can get a multiple alignment from a group of related protein sequences using the Block Maker or other automated methods(such as ClustalW). The alignments can also be manually made or modified according to your knowledge of the proteins (position of the active sites orpost-translational modifications etc.). In any case the alignments passed to the CODEHOP program must be in the Blocks format. BlockMaker blocks need no reformatting. Appropriate parts of Clustal- or FASTA-formatted multiple alignments can be automatically made into blocks by the Blocks multiple alignment processor. Other types of multiple alignment can be semi-manually reformatted with the Blocks formatter. All of the above Blocks programs have links to send the resulting blocks directly to theCODEHOP program and also provide other information for evaluating the blocks (logos, trees, searches).More information on multiple alignments can be found in the notes for the ISMB97 tutorial on Introduction to making and using protein multiple alignments.Terms and parameters•Degeneracy measures how many different sequences are specified by the primer. The degeneracy of each position depends on the sum of the nucleotides appearing in it (1 to 4). The degeneracy of the primer is the product of the degeneracies of all of its positions.The degenerate core region is scored by its degeneracy, with lower degeneracy scored higher. A standard degenerate alphabet is used in the core region.•Degeneracy strictness specifies how to count nucleotide(s) with low occurrences. It is a parameter between 0 and 1. Strictness of 0 means that all nucleotides that actually appear in the position arecounted to calculate the degeneracy. Strictness of 1 means that only the nucleotides with the highest value in the position are counted.Intermediate strictness values give behavior in between. You can look at a detailed explanation for more information on how thestrictness is calculated.•Temperature is computed for the clamp region plus any adjacent non-degenerate positions in the core region. Differenttemperatures affect only the length of the clamp; highertemperatures result in longer clamps. Temperature is computed using the nearest-neighbor method of Rychlik, et al ( NAR 1990 18:21, 6409-6412). This method takes into consideration the primer and K+ (potassium ions, taken as 50 mM) concentrations.•Nucleotide runs in the clamp region can be limited (the default is5 in a row). If longer runs are encountered, they are broken up bytaking the next highest- scoring nucleotide in the position with the lowest-scoring run nucleotide.•The clamp score is a measure of how well the non-degenerate 5' clamp matches the block given a codon usage table. The score is a weighted average of the maximum scores in each column of the DNA PSSM. A perfect score of 100 is obtained if the clamp consists of invariant methionines and tryptophans, and a minimum score of 25 is obtained if A, C, G and T are equally likely at all positions. In computing the clamp score, positions are increasingly down-weighted asdistance from the degenerate 3' core increases. The clamp score is intended to estimate how well the clamp will stabilize the core inannealing to the target templates. All else being equal, the higher the clamp score the better. However, the clamp score is not relevant to stability of the clamp/product hybrid, which is estimated by the annealing temperature using the nearest neighbor method.•The core/clamp boundary can be restricted to always be on a codon boundary.•Genetic code tables are used to back-translate from protein to DNA.•Codon usage tables are derived from the Codon Usage Database.Tables currently available: request tables for other organisms here.The codon usage table is used to convert the amino acid PSSMconstructed from the input blocks into the DNA PSSM. It can also be used to determine the content of the clamp region.•Clamp residues can be selected as the most common codons of the consensus amino acids are used in the clamp. Else the clamp residues are the ones with maximum weight in the DNA PSSM, which may result in 'artificial' codons. If the clamp residues are taken from the DNA PSSM and more than one residue in a position have maximum weight, one of them is selected at random to keep the clamp non-degenerate.•The program output can show all possible primers or only the single most degenerate primer in each region.Basic tipsOnce you have a block(s) in the input window of the CODEHOP page you can "Look for primers" using the default parameters. You can adjust the setting according to your intended use of the primers and the results you got with the defaults. If you don't get predictions, or you don't like what you get we recommend to first raise the degeneracy to 256 or higher (if you dare ...) and retry. Next, you might try raising the strictness of the core region, for example to 0.1 or 0.25.Your target sequence(s) might be expected to be more similar to some specific sequence(s) in the input blocks. In this case you can bias the primers towards these sequences. Rather than raising the degeneracy or strictness, increase the weights of the specific sequences. The weights are the values following each sequence segment of the block. Usually the highest weight is 100. In the CODEHOP input window increase the weights of your specific sequences (say to 3 or 4 times the original weight or to 200 or 400). You can also remove individual sequences from the input blocks by down-weighing them to 0 (the minimal weight) if they are too divergent and prevent finding primers.For amplification, we recommend using AmpliTaq Gold with a 9' preheat (this provides an automatic hotstart - a hotstart of some kind isimportant). We have had success using the time-release feature with the addition of 15-20 extra cycles. The primer finding strategy of the CODEHOP program (Rose, et al, manuscript accepted by NAR) is different from the usual degenerate PCR strategies. It is desirable to keep annealing temperatures high - 60o C is OK if you have a 60o C clamp. We recommend trying the highest temperature that yields a clean PCR product. We have used "touchdown" PCR down to Tm-3o C or lower, say from 63o C down to a good clamp annealing temperature in -0.5 to -1o C increments, and the remaining cycles are carried out at the 53-57o C clamp annealing temperature for a 60o C clamp. The intent of the touchdown is to give the correct product a head start, because it is likely to anneal at a higher temperature than any failure product. Once the clamp 'takes over', then all primed products, whether correct or not, will be on an even footing, so we try to keep the stringency high in all cycles. With luck, it should not be necessary to gel-purify product, but may rather try cloning directly from the reaction mix if a single band of the expected size is obtained.We and a few other users were already successful in using the CODEHOP strategy and program to amplify various sequences from complicated and diverged genomes. Please let us know if you have any tips to pass on based on your experience using CODEHOP-predicted primers.PublicationResults obtained by this method should cite:"Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences" by T.M. Rose, E.R. Schultz, J.G. Henikoff, S. Pietrokovski, C.M. McCallum and S. Henikoff, Nucleic Acids Research, 26(7):1628-1635.Genes identified using CODEHOP.CODEHOP programThe CODEHOP program designs a pool of primers containing all possible 11- or 12-mers for the 3' degenerate core region and having the most probable nucleotide predicted for each position in the 5' non-degenerate clamp region.The program consists of the following steps: (note the scheme on the right)1) A set of blocks is input, where a block is an aligned array of amino acidsequence segments without gaps that represents a highly conserved region ofhomologous proteins. A weight is provided for each sequence segment, which can beincreased to favor the contribution of selected sequences in designing the primer.A codon usage table is chosen for the target genome.2) An amino acid position-specific scoring matrix (PSSM) is computed for each blockusing the odds ratio method.3) A consensus amino acid residue is selected for each position of the block as thehighest scoring amino acid in the matrix.4) For each position of the block, the most common codon corresponding to the aminoacid chosen in step 3 is selected utilizing the user-selected codon usage table.This selection is used for the default 5' consensus clamp in step 8.5) A DNA PSSM is calculated from the amino acid matrix (step 2), genetic codetable and codon usage table. The DNA matrix has three positions for each position of the amino acid matrix. The score for each amino acid is divided amongits codons in proportion to their relative weights from the codon usage table, andthe scores for each of the four different nucleotides are combined in each DNAmatrix position. Nucleotide positions are treated independently when the scores arecombined. As an option, the highest scoring nucleotide residue from each positioncan replace the most common codons from step 4 that are used in the consensus clamp.6) The degeneracy is determined at each position of the DNA matrix based on thenumber of bases found there. As an option, a weight threshold can be specified suchthat bases that contribute less than a minumum weight are ignored in determiningdegeneracy.7) Possible degenerate core regions are identified by scanning the DNA matrix inthe 3' to 5' direction. A core region must start on an invariant 3' nucleotideposition, have length of 11 or 12 positions ending on a codon boundary, and have amaximum degeneracy of 128 (current default). The degeneracy of a region is theproduct of the number of possible bases in each position.8) Candidate degenerate core regions are extended by addition of a 5' consensus clampfrom step 4 or 5. The length of the clamp is controlled by a melting point temperaturecalculation (current default = 60o) and is usually ~20 nucleotides.9) Steps 7 and 8 are repeated on the reverse complement of the DNA matrix from step5 for primers corresponding to the opposite DNA strand.CODEHOP program scheme1) input - - - - - - - - - - - - - - - seq 1 Protein sequence block - - - - - - - - - - - - - - - seq 2- - - - - - - - - - - - - - - seq 3- - - - - - - - - - - - - - - seq 4- - - - - - - - - - - - - - - seq 5- - - - - - - - - - - - - - - etc.|| 2) transformation to AA PSSMV| | | | | | | | | | | | | | | Ala AA PSSM| | | | | | | | | | | | | | | Cys| | | | | | | | | | | | | | | Asp| | | | | | | | | | | | | | | Glu| | | | | | | | | | | | | | | Phe| | | | | | | | | | | | | | | Gly| | | | | | | | | | | | | | | His| | | | | | | | | | | | | | | Ile| | | | | | | | | | | | | | | Lys| | | | | | | | | | | | | | | etc.| || | 3) calculation of AA consensus sequence| V| - - - - - - - - - - - - - - - AA consensus sequence || || | 4) transformation to DNA consensus sequence| V| ------------------------------------------- DNA consensus sequence | || | 5) back-translation to DNA PSSM| V| ||||||||||||||||||||||||||||||||||||||||||| A DNA PSSM| ||||||||||||||||||||||||||||||||||||||||||| C| ||||||||||||||||||||||||||||||||||||||||||| G| ||||||||||||||||||||||||||||||||||||||||||| T| | || | | 6) calculation of degeneracies| | V| | ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ position degeneracy values | || | 7) identify degenerate regions ("===")| || 8) identify consensus regions for degenerate regions ("---")| |V V5' -------==== 3' CODEHOP primers output 3' ====--------- 5'。