Computational Biology, Part 5 PCR Primer Design & Basic Principles of Entrez Robert F. Murphy Copyright 1996, 1999, 2000, 2001. All rights reserved

Computational Biology, Part 5PCR Primer Design &

Basic Principles of Entrez

Computational Biology, Part 5PCR Primer Design &

Basic Principles of Entrez

Robert F. MurphyRobert F. Murphy

Copyright Copyright 1996, 1999, 2000, 2001. 1996, 1999, 2000, 2001.

All rights reserved.All rights reserved.

Polymerase Chain ReactionPolymerase Chain Reaction

Method for exponential amplification of Method for exponential amplification of DNA or RNA sequencesDNA or RNA sequences

Basic requirementsBasic requirements templatetemplate DNA or RNA DNA or RNA 2 oligonucleotide 2 oligonucleotide primersprimers complementary to complementary to

different regions of the templatedifferent regions of the template heat stable heat stable DNA polymeraseDNA polymerase 4 nucleotides and appropriate buffer4 nucleotides and appropriate buffer

PCR reference booksPCR reference books

PCR Protocols: A Guide to Methods and PCR Protocols: A Guide to Methods and Applications. Applications. M. A. Innis, D. H. Gelfand, J. J. M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White (eds.) Sninsky, T. J. White (eds.) Academic Press, Inc., San Academic Press, Inc., San Diego, 1990Diego, 1990

PCR: A Practical Approach. PCR: A Practical Approach. M. J. M. J. McPherson, P. Quirke, G. R. Taylor (eds.) McPherson, P. Quirke, G. R. Taylor (eds.) Oxford Oxford University Press, Oxford, 1991University Press, Oxford, 1991

Basic Principles of PCRBasic Principles of PCR

1. Strands of 1. Strands of templatetemplate DNA (or RNA) are separated by DNA (or RNA) are separated by meltingmelting

2. 2. ForwardForward PrimerPrimer binds to one strand of template, binds to one strand of template, Reverse Primer Reverse Primer to other strandto other strand

3. 3. DNA polymerase DNA polymerase extends 3’ end of each primer, extends 3’ end of each primer, copying templatecopying template

4. Strands are separated by raising temperature, allowing 4. Strands are separated by raising temperature, allowing both original DNA and copies to act as templatesboth original DNA and copies to act as templates

5. Repeat steps 2-4 many times5. Repeat steps 2-4 many times

Temperature cyclingTemperature cycling

Annealing temperature Annealing temperature (usually 45-60(usually 45-60C) C) allows primers to hybridize to templateallows primers to hybridize to template

Extension temperature Extension temperature (usually 72(usually 72C) C) allows polymerase to extend starting at the allows polymerase to extend starting at the primerprimer

Denaturation temperature Denaturation temperature (usually 95(usually 95C) C) separates strandsseparates strands

PCRPCR

Heat

Heat

Cool

Cool

Heat etc.

2 copies2 copies

+ and - strands of templateforward primerreverse primeramplified DNA 3’

5’

5’

3’5’

3’

3’

5’

4 copies4 copies

8 copies8 copies

Heat

Cool

add primers, polymerase, dNTPs

1 copy1 copy

Primer Design ConsiderationsPrimer Design Considerations

Primers must be specific for desired sequence to Primers must be specific for desired sequence to be amplifiedbe amplified primers should be long enough to ensure specificity primers should be long enough to ensure specificity

(usually 18-30 bases)(usually 18-30 bases) primers normally screened against databasesprimers normally screened against databases

Primers must form stable duplex at annealing Primers must form stable duplex at annealing temperaturetemperature

No complementarity between forward and No complementarity between forward and reverse primers or primers and productreverse primers or primers and product

Initial primer selection criteriaInitial primer selection criteria

Length (18-25 bases)Length (18-25 bases) Base composition (45-55% GC)Base composition (45-55% GC) Melting temperature (55-80Melting temperature (55-80C)C) 3’ terminal sequence3’ terminal sequence

strong bonding base (G or C) at endstrong bonding base (G or C) at end no runs (3 or more) of G or C at endno runs (3 or more) of G or C at end

Primer complementarity criteriaPrimer complementarity criteria

Primer vs. self & forward vs. reversePrimer vs. self & forward vs. reverse maximum number of consecutive bondsmaximum number of consecutive bonds maximum number of consecutive G-C bondsmaximum number of consecutive G-C bonds

Forward primer vs. Reverse primerForward primer vs. Reverse primer maximum number of consecutive bonds between the maximum number of consecutive bonds between the

3’ ends3’ ends Primer vs. productPrimer vs. product

maximum number of consecutive bonds between the maximum number of consecutive bonds between the 3’ ends3’ ends

Optimization criteriaOptimization criteria

Melting temperatures should be similar for Melting temperatures should be similar for both primersboth primers

Product should be as short as allowableProduct should be as short as allowable

Automated PCR probe selection References


W. Rychlik & R. E. Rhoads. A computer program for W. Rychlik & R. E. Rhoads. A computer program for choosing optimal oligonucleotides for filter hybridization, choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. sequencing and in vitro amplification of DNA. Nucleic Nucleic Acids Res 17:Acids Res 17:8543-51 (1989)8543-51 (1989)

A method is presented for choosing optimal oligodeoxyribonucleotides as probes for A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4 degrees C of the values obtained predicted by the method described here were within 0.4 degrees C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.self-complementarity are based on a simple dynamic algorithm.



T. Lowe, J. Sharefkin, S. Q. Yang & C. W. Dieffenbach. T. Lowe, J. Sharefkin, S. Q. Yang & C. W. Dieffenbach. A computer program for selection of oligonucleotide A computer program for selection of oligonucleotide primers for polymerase chain reactions. primers for polymerase chain reactions. Nucleic Acids Res Nucleic Acids Res 18:18:1757-61 (1990)1757-61 (1990)

We have designed a computer program which rapidly scans nucleic acid sequences to We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions while providing a flexible choice of various primers to focus study on particular regions of interest.of interest.



L. Hillier & P. Green. OSP: a computer program for choosing L. Hillier & P. Green. OSP: a computer program for choosing PCR and DNA sequencing primers. PCR and DNA sequencing primers. PCR Methods Appl PCR Methods Appl 1:1:124-8 (1991)124-8 (1991)

OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA sequencing OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA sequencing and the polymerase chain reaction (PCR). The user can specify (or use default) constraints for and the polymerase chain reaction (PCR). The user can specify (or use default) constraints for primer and amplified product lengths, %(G+C), (absolute or relative) melting temperatures, primer and amplified product lengths, %(G+C), (absolute or relative) melting temperatures, and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary structure, OSP screens candidate primer sequences, using user-specifiable cutoffs, against structure, OSP screens candidate primer sequences, using user-specifiable cutoffs, against potential base-pairing with a variety of sequences present in the reaction, including the primer potential base-pairing with a variety of sequences present in the reaction, including the primer itself, the other primer (for PCR), the amplified product, and any other sequences desired (e.g., itself, the other primer (for PCR), the amplified product, and any other sequences desired (e.g., repetitive element sequences in genomic templates, vector sequence in cloned templates, or repetitive element sequences in genomic templates, vector sequence in cloned templates, or other primer pair sequences in multiplexed PCR reactions). Base-pairing involving the primer other primer pair sequences in multiplexed PCR reactions). Base-pairing involving the primer 3' end is considered separately from base-pairing involving internal sequences. Primers 3' end is considered separately from base-pairing involving internal sequences. Primers meeting all constraints are ranked by a "combined score," a user-definable weighted sum of meeting all constraints are ranked by a "combined score," a user-definable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the Caenorhabditis elegans genome sequencing project and human genomic PCR primers for the Caenorhabditis elegans genome sequencing project and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 96% and 81%, respectively. It is available for research purposes from the rates exceeding 96% and 81%, respectively. It is available for research purposes from the authors, at no cost, in both text output and interactive graphics (X windows) versions.authors, at no cost, in both text output and interactive graphics (X windows) versions.

List-based rule applicationList-based rule application

Define rulesDefine rules Select initial list of itemsSelect initial list of items Select subsets of list that pass each rule, Select subsets of list that pass each rule,

keeping count of number of times that each keeping count of number of times that each reason is used to reject an itemreason is used to reject an item

Finding PCR primers with MacVectorFinding PCR primers with MacVector Two search typesTwo search types

specify product size within a regionspecify product size within a region example: detection of gene in a DNA or RNA example: detection of gene in a DNA or RNA

samplesample

specify two flanking regionsspecify two flanking regions example: subcloning of a plasmid fragment or insertexample: subcloning of a plasmid fragment or insert

Two search typesTwo search types

IllustrationIllustration

Goal: Find primers for detecting presence of Goal: Find primers for detecting presence of tubulin exon 5 in mRNAtubulin exon 5 in mRNA


Specify Region to scan and use defaults for Specify Region to scan and use defaults for all other parametersall other parameters


Conclusion: Too many pairs foundConclusion: Too many pairs found

IllustrationIllustration Be more restrictive: Require strong 3’ Be more restrictive: Require strong 3’

anchoranchor


Conclusion: Still too manyConclusion: Still too many


Reduce allowed bonding to product alsoReduce allowed bonding to product also


8 is few enough. Request graphical map.8 is few enough. Request graphical map.


Can Can make make final final choice choice based based on on product product sizesize

What if no primers are found?What if no primers are found?

Examine display listing number of primers or Examine display listing number of primers or pairs rejected by each criterionpairs rejected by each criterion

Loosen appropriate criterionLoosen appropriate criterion If few forward or reverse primers are being If few forward or reverse primers are being

accepted, broaden primer length, end base accepted, broaden primer length, end base restriction, Trestriction, Tmm limits, or %GC limits limits, or %GC limits

If no primer pairs accepted, loosen primer vs. primer If no primer pairs accepted, loosen primer vs. primer or primer vs. product criteriaor primer vs. product criteria

Repeat searchRepeat search

Last stepLast step

Compare primer sequences against nucleic Compare primer sequences against nucleic acid sequence databasesacid sequence databases

Calculate TCalculate Tmm for duplex between primer and for duplex between primer and

best match foundbest match found Compare with TCompare with Tmm of primer itself of primer itself

Web sites related to PCRWeb sites related to PCR

PCR primer designer: PCR primer designer: Primer3Primer3 http://www-genome.wi.mit.edu/cgi-http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgibin/primer/primer3.cgi

All you ever wanted to know about PCRAll you ever wanted to know about PCR http://www.apollo.co.uk/a/pcrhttp://www.apollo.co.uk/a/pcr

Downloadable PCR design software catalogDownloadable PCR design software catalog http://bioinformatics.weizmann.ac.il/http://bioinformatics.weizmann.ac.il/mb/bioguide/pcr/software.htmlmb/bioguide/pcr/software.html

Designing primers for sequence familiesDesigning primers for sequence families Most PCR primer design software considers Most PCR primer design software considers

only one template sequenceonly one template sequence Different software is required whenDifferent software is required when

designing a single set of primers to amplify more designing a single set of primers to amplify more than one template sequencethan one template sequence

designing a set of primers that will amplify only a designing a set of primers that will amplify only a specific member of a set of sequencesspecific member of a set of sequences

Examples:Examples: detecting all members of a gene familydetecting all members of a gene family detecting a highly variable gene, such as a viral genedetecting a highly variable gene, such as a viral gene

Designing primers for families References


K. Lucas, M. Busch, S. Mossinger & J. A. Thompson. An K. Lucas, M. Busch, S. Mossinger & J. A. Thompson. An improved microcomputer program for finding gene- or gene improved microcomputer program for finding gene- or gene family-specific oligonucleotides suitable as primers for family-specific oligonucleotides suitable as primers for polymerase chain reactions or as probes. polymerase chain reactions or as probes. Comput Appl Biosci Comput Appl Biosci 7:7:525-9 (1991)525-9 (1991)

We present here an easy-to-use computer program which finds oligonucleotides suitable as We present here an easy-to-use computer program which finds oligonucleotides suitable as primers in polymerase chain reactions (PCR) or as probes for hybridization. In contrast to primers in polymerase chain reactions (PCR) or as probes for hybridization. In contrast to other programs used for this purpose, the additional advantage of this one is the possibility of other programs used for this purpose, the additional advantage of this one is the possibility of directly detecting gene-as well as gene family-specific oligonucleotides. For this purpose, up directly detecting gene-as well as gene family-specific oligonucleotides. For this purpose, up to 200 different DNA sequences, of maximally 65,000 nucleotides each, can be scanned in a to 200 different DNA sequences, of maximally 65,000 nucleotides each, can be scanned in a single search to ensure either single or multiple gene binding of the PCR primers or probes. single search to ensure either single or multiple gene binding of the PCR primers or probes. Specific oligonucleotides for genes carrying internal repetitions and for single genes Specific oligonucleotides for genes carrying internal repetitions and for single genes belonging to a set of highly conserved genes can also be detected. Many parameters such as belonging to a set of highly conserved genes can also be detected. Many parameters such as exclusion of simple sequences, which are known to be highly repeated throughout various exclusion of simple sequences, which are known to be highly repeated throughout various genomes or regions of stable secondary structures in both primer-primer and primer-template, genomes or regions of stable secondary structures in both primer-primer and primer-template, can be taken into consideration and avoided. Furthermore, the G + C content and the length can be taken into consideration and avoided. Furthermore, the G + C content and the length of the oligonucleotides can be changed in a broad range by the user.of the oligonucleotides can be changed in a broad range by the user.



M. L. Montpetit, S. Cassol, T. Salas & M. V. O'Shaughnessy. M. L. Montpetit, S. Cassol, T. Salas & M. V. O'Shaughnessy. OLIGSCAN: a computer program to assist in the design of OLIGSCAN: a computer program to assist in the design of PCR primers homologous to multiple DNA sequences. PCR primers homologous to multiple DNA sequences. J J Virol Methods 36:Virol Methods 36:119-28 (1992)119-28 (1992)

OLIGSCAN (oligonucleotide scanner) is a computer program for IBM-PC-compatible OLIGSCAN (oligonucleotide scanner) is a computer program for IBM-PC-compatible computers that allows the user to scan up to 200 DNA sequences for homology to computers that allows the user to scan up to 200 DNA sequences for homology to oligonucleotide sequences of interest. Once a core sequence of longer than the user-defined oligonucleotide sequences of interest. Once a core sequence of longer than the user-defined minimum length is found, the remainder of the oligonucleotide is compared to the minimum length is found, the remainder of the oligonucleotide is compared to the corresponding positions of the larger sequence to identify matches or mismatches flanking corresponding positions of the larger sequence to identify matches or mismatches flanking the core region. This algorithm results in identification of the longest possible homologous the core region. This algorithm results in identification of the longest possible homologous regions first. The program was originally designed to assist in the identification of potential regions first. The program was originally designed to assist in the identification of potential annealing sites for polymerase chain reaction (PCR) primers in the genomic DNA of related annealing sites for polymerase chain reaction (PCR) primers in the genomic DNA of related strains of viruses. However, it may also be used for more general pattern-identification strains of viruses. However, it may also be used for more general pattern-identification purposes, including scanning for various sequence motifs of functional importance. We purposes, including scanning for various sequence motifs of functional importance. We present the analysis of homology to an oligonucleotide primer in 16 complete genomic present the analysis of homology to an oligonucleotide primer in 16 complete genomic sequences of the human and simian immunodeficiency viruses.sequences of the human and simian immunodeficiency viruses.



J. Dopazo & F. Sobrino. A computer program for the J. Dopazo & F. Sobrino. A computer program for the design of PCR primers for diagnosis of highly variable design of PCR primers for diagnosis of highly variable genomes. genomes. J Virol Methods 41:J Virol Methods 41:157-65 (1993)157-65 (1993)

PCRDiag (Diagnosis by PCR) is a computer program which allows the localization of PCRDiag (Diagnosis by PCR) is a computer program which allows the localization of pairs of oligonucleotides with optimal thermodynamic requirements for use in a PCR pairs of oligonucleotides with optimal thermodynamic requirements for use in a PCR assay. The program is designed for the selection of pairs of primers complementary to assay. The program is designed for the selection of pairs of primers complementary to sequences present in a group, whose identification is intended, but are absent in other sequences present in a group, whose identification is intended, but are absent in other non-specific sequences. The program constitutes a powerful tool, specially in systems non-specific sequences. The program constitutes a powerful tool, specially in systems which display a high degree of sequence heterogeneity, as is the case of RNA viruses. which display a high degree of sequence heterogeneity, as is the case of RNA viruses. The program runs on IBM-PC and compatible computers and has no special software The program runs on IBM-PC and compatible computers and has no special software requirements. It does not need the previous alignment of the sequences analyzedrequirements. It does not need the previous alignment of the sequences analyzed

Web sites for degenerate primersWeb sites for degenerate primers

List of various PCR primer design web sitesList of various PCR primer design web sites http://www.hgmp.mrc.ac.uk/http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.htmlGenomeWeb/nuc-primer.html

PCR degenerate primer designer for PCR degenerate primer designer for multiple sequencesmultiple sequences http://bibiserv.techfak.uni-bielefeld.de/http://bibiserv.techfak.uni-bielefeld.de/genefisher/genefisher/

Block Diagram for PCR Primer DesignBlock Diagram for PCR Primer Design

PCR Primer List Processor

PCR reaction parameters

Sequence from which to choose primers

Primer Selection Rules

Results of Search, including suggested annealing temperatures (List)

Network EntrezNetwork Entrez

a client-server system for retrieval of a client-server system for retrieval of information related to molecular biologyinformation related to molecular biology

can be usedcan be used via web pagevia web page via local copy of Entrez clientvia local copy of Entrez client via "embedded" client in other software (e.g., via "embedded" client in other software (e.g.,

MacVector)MacVector)

Entrez DatabasesEntrez Databases

LiteratureLiterature PUBMED database contains Medline abstracts as well as links PUBMED database contains Medline abstracts as well as links

to full text articles on sites maintained by journal publishersto full text articles on sites maintained by journal publishers

Nucleic acid sequencesNucleic acid sequences Protein sequencesProtein sequences 3D structures3D structures GenomesGenomes TaxonomyTaxonomy

Entrez literature searchingEntrez literature searching

can find papers on a given subjectcan find papers on a given subject can find papers on a specific genecan find papers on a specific gene can find papers related to a given papercan find papers related to a given paper can switch between literature and sequence can switch between literature and sequence

databasesdatabases

Entrez sequence searchingEntrez sequence searching

can find sequences for a given genecan find sequences for a given gene can download copy of sequencecan download copy of sequence

Example Entrez SessionExample Entrez Session

Goal: Find literature and sequences for Goal: Find literature and sequences for cystic fibrosis genescystic fibrosis genes Use Use Nucleotide DatabaseNucleotide Database with with KeywordKeyword

searching.searching.


Goal: Find literature and sequences for Goal: Find literature and sequences for cystic fibrosis genescystic fibrosis genes Use Use MEDLINEMEDLINE with with KeywordKeyword searching. searching. Use Use neighborneighbor feature to find related articles. feature to find related articles.


Goal: Find literature and sequences for Goal: Find literature and sequences for obesity genesobesity genes Use Use MEDLINEMEDLINE with with KeywordKeyword searching. searching. Use Use neighborneighbor feature to find related articles. feature to find related articles. Switch to Switch to NucleotideNucleotide database to see sequence. database to see sequence.


Goal: Find literature and sequences for Goal: Find literature and sequences for obesity genesobesity genes Use Use MEDLINEMEDLINE with with KeywordKeyword searching. searching. Use Use neighborneighbor feature to find related articles. feature to find related articles. Switch to Switch to NucleotideNucleotide database to see sequence. database to see sequence. Save a copy of sequence to local disk.Save a copy of sequence to local disk.


Goal: Find literature and sequences for Goal: Find literature and sequences for obesity genesobesity genes Use Use MEDLINEMEDLINE with with KeywordKeyword searching. searching. Use Use neighborneighbor feature to find related articles. feature to find related articles. Switch to Switch to NucleotideNucleotide database to see sequence. database to see sequence. Save a copy of sequence to local disk.Save a copy of sequence to local disk. Use Use MESHMESH terms to find similar articles. terms to find similar articles.


Goal: Find literature and sequences for Goal: Find literature and sequences for obesity genesobesity genes Use Use MEDLINEMEDLINE with with KeywordKeyword searching. searching. Use Use neighborneighbor feature to find related articles. feature to find related articles. Switch to Switch to NucleotideNucleotide database to see sequence. database to see sequence. Change to Change to GenbankGenbank format to save sequence. format to save sequence. Use Use MESHMESH terms to find similar articles. terms to find similar articles. Search the Search the NucleotideNucleotide database by database by genegene name. name.

Suggested Entrez ExplorationsSuggested Entrez Explorations

Find the sequence of the Find the sequence of the ArabidopsisArabidopsis ACT1 ACT1 mRNA.mRNA.

Find the classic article on cloning of full-Find the classic article on cloning of full-length cDNA by H. Okayama and P. Berg. length cDNA by H. Okayama and P. Berg.

Block Diagram for Entrez Literature SearchingBlock Diagram for Entrez Literature Searching

Entrez Search Engine

Additional Search Criterion

Desired Output Format

Results of Previous Search

Displayed Item Selection

Results of Search (List)

Item Display

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Computational Biology, Part 5 PCR Primer Design & Basic Principles of Entrez Robert F. Murphy Copyright 1996, 1999, 2000, 2001. All rights reserved