Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
OSP: A Computer Program for Choosing PCR and DNA
Sequencing Primers LaDeana Hillier and Philip Green Genetics Department, Washington University School of Medicine St. Louis, Missouri 63110
OSP (Oligonucleotide Selection Program) selects ollgonucleotide primers for DNA sequencing 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, and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary 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 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 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 meeting all constraints are ranked by a "combined score," a userdefinable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the Caenorhabditis elegons genome sequencing project and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 9 6 % 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. 124 PCR Methods and Applications
DNA sequencing by the Sanger chain-termination method, (1) and the polymerase chain reaction (PCR), (2) are ubiquitous molecular biology tools that require oligonucleotides for priming DNA synthesis. Not all primers work, and those that do vary widely in their resistance to changes in reaction conditions, a situation that has spurred the development of several computer programs for choosing primers. (3,4) The onset of major mapping and sequencing efforts as part of the Genome Initiative has increased the demand for efficient primer selection. OSP (Oligonucleotide Selection Program) was designed to satisfy the needs of two projects: the Washington University Genome Center's project to map human chromosomes 7 and X, which will require PCR assays to detect well over 1000 sequence-tagged sites (STSs) (s) in h u m a n genomic DNA, and
the Caenorhabditis elegans genome sequencing project, which is presently using a mixed shotgun sequencing and walking strategy requiring selection of sequencing primers (roughly 100 per cosmid) for the walking steps. Major requirements for OSP were ease of use and flexibility in setting primer selection criteria, so that the program could evolve in the light of experience gained in both projects. In this paper we describe O S P , and summarize results obtained using it.
METHODS OSP: Program Design and Operation Input of sequences For selecting PCR primers, the user provides either one sequence that includes the region to be amplified or two sequences flanking that region; for selec-
ting sequencing primers, a single sequence must be provided. Either strand may be given, and ambiguous nucleotides within the sequence are permitted (although primers containing ambiguous nucleotides are rejected). Sequences may either be entered at run time (in the graphics version of the program, they may be "pasted" in from another window) or provided as a text file(s) in a variety of possible formats. The user may confine the search for candidate primers to particular regions of the input sequence. The primer locations may also be specified exactly, if the user wishes to compute scores or other characteristics for previously selected primers.
Constraints Primer and amplified product characteristics that may be constrained are listed in Table 1, along with OSP's default values, which have been found to work well in several mapping and sequencing projects (see Results). The constraint criteria are those thought (largely on the basis of anecdotal evidence) to be important to ensure sensitive and specific priming: for example, if G+C content is too high, the primer may be more prone to adopt secondary structure or to anneal nonspecifically to GC-rich regions of the template DNA, while if it is too low the primer may not anneal to its target sequence. If the user does not wish to use the defaults, modified constraint values for any or all parameters may be provided in a "constraint file," and/or entered directly at run time (the constraints for any parameter can also be disabled). "Annealing scores" are intended to provide simple measures of basepairing propensity of the primer with
1:124-128@1991 by Cold Spring Harbor Laboratory PressISSN1054-9803/91 $3.00
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
TABLE 1 User-constrainable Parameters, Default Limits, a n d Default Weights
Minimum
Maximum
Weight
Single primer parameters 3 ' nucleotidesa Primer length (nucleotides)c Primer G+C content (%) Primer Tm (Celsius) d Annealing scorese Primer-self internal Primer-self 3' Primer-other internal Primer-other 3 '
Sa
18 40.0 50.5 --
mb
22 55.0 55.0
0.0 0.0 0.0
14.0
1.0
--
8.0 NSf
2.0 0.0
--
NS
0.0
--
Primer pair parameters Product length (bp)g Product G÷C content (%) Product Tm (Celsius) Primer Tm difference (Celsius) Annealing scores Primer-primer internal Primer-primer 3 ' Primer-product internal Primer-product 3'
100 40.0 70.0 --
D
300 $5.0 90.0 2.0
0.0 0.0 0.0 0.0
14.0 8.0 NS NS
1.0 2.0 0.0 0.0
aAny string of 3 ' terminal nucleotide(s) for the primers may be specified, with all standard ambiguity codes allowed. The default is a single nucleotide S, which matches either C or G. b(--)Not meaningful. CValues shown are for PCR primers. For sequencing primers, defaults are rain. = 17, max. = 18. dTm (melting temperature) is calculated as: 62.3 + 0.41(%(G+C)) - 500/length. (6) Any ambiguous nucleotides are ignored. eSee text for explanation. f(NS) Not set (in OSP defaults). gFor sequencing primers, product length is the distance from the end of the sequence. Default constraints are min. = 40, max. = 100. the various n o n t a r g e t sequences present in the reaction. They are c o m p u t e d as follows: The user may define a score for an A/T base pair (default value, 2) and for a C/G base pair (default value, 4). For a n y a l i g n m e n t of two sequences (with the first sequence written 5 ' t o 3 ' , and the second one 3 ' t o 5 ' ) scores are s u m m e d over contiguous c o m p l e m e n t a r y nucleotide pairs; the m a x i m u m such score, taken over all contiguous blocks in all alignments, is the internal a n n e a l i n g score. The 3 ' a n n e a l i n g score (which is c o m p u t e d and weighted separately because it likely has a greater influence on nonspecific priming) is based only o n contiguous c o m p l e m e n t a r y matches that include the 3 ' e n d of the primer. Annealing scores are calculated for the primer with itself, with the other primer, with the amplified product, a n d with a user supplied set of "other" sequences. At a m i n i m u m , the latter would typically include repetitive sequences w h e n the template is g e n o m i c
DNA, or vector sequence w h e n the template is cloned DNA. It could also include other primer sequences if one is interested in "multiplexing" multiple PCR primer pairs in the same reaction tube. For example, the a l i g n m e n t 5 ' -ACGGATTGTGCCGTATTG-3 ' 3' TGACACGTGTAGACGAGG-5 ' of two candidate primers would yield an internal a n n e a l i n g score of 16 (using the default base-pair scores), corresponding to the pairing of TGTGC on the top strand with ACACG o n the bottom, and a 3 ' a n n e a l i n g score of 6, corresponding to the pairing of TG at the 3 ' e n d of the first primer with AC. Since the default internal a n n e a l i n g cutoff (14.0) is exceeded, this pair would be rejected by OSP.
position w i t h i n the sequence, it tests candidate primers h a v i n g that position as the 3 ' terminus, e x a m i n i n g in order the criteria in the upper half of Table 1. Primers c o n t a i n i n g an ambiguous nucleotide are automatically rejected. Primers with the same 3 ' e n d are considered in order of increasing length; this allows consideration of longer primers to be aborted w h e n " m o n o t o n ically increasing" parameters (e.g., primer-self or primer-other annealing) exceed their constraint values. If all criteria are satisfied, the primer is saved in a list of candidates for that strand.
PCR Primer Pair Selection Algorithm Following construction of lists of candidate primers for each strand, candidate primer pairs (one from each list) are considered. If several pairs have the same 3 ' ends for b o t h primers, at most one pair (the shortest) is accepted. For each candidate pair, constraints involving the amplified product and primer pair (lower half of Table 1) are tested, and any pair meeting each applicable constraint is added to the list of valid pairs. Once all possible primer pairs have been considered, t h e y are ranked based on their "combined score," w h i c h is a user-definable weighted sum of a n y of the parameters in Table 1. The default c o m b i n e d score (which uses the default weights in Table 1) is based o n the primer-self and primer-primer annealing scores, a n d weighs 3 ' annealing twice as m u c h as internal annealing. The user m a y supply different weights in the constraint file.
Program Output OSP notifies the user of the n u m b e r of primers (or primer pairs) accepted, a n d provides a table listing the n u m b e r of pairs rejected for each constraint. (This is often useful in cases where n o pair meets all constraints.) The user can t h e n choose to save (to a file) a ranked list of as m a n y of the primers as desired, along with their characteristics. The o u t p u t file also records the constraint values used and the n u m bers of primers or pairs accepted and rejected.
Primer Selection Algorithm OSP scans each strand in a 5 ' t o 3 ' direction for possible primers. For each
Graphic Interface A
menu-driven
"point
and
click"
PCR Methods and Applications 125
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
~lick
TYPE/PASTE IN SEQUENCE uiLh the 1 e l l eouse button ON any primer to have score i n f e r e a t i o n displayed here
217
PRODUCT CONSTRAINTS Min
G.c Conte.t(~):
.a. 130~ .in L4~ Na, [5t
) ]
min
1
Tn(Celsius):
.a.
~IC.NCEL
[10~
170. ~ [90.0
]
Length(bases):
Min
118
]
Ha.
12~
I
Min
140
]
.a.
155.
[
Min
150. ~
J
.a.
155.~
I
Difference in Te cuLoFF:
I 2.0
l
3" NucleoLides (S=G/C,g=A/T):
IS~
I
l G+C ConLent(X);
Tm(Celslus):
J
279
310
341
372
403
)RIMERS (Nunber o£ S-type sequences Found uas 183) Sense: T o t a l considered: Rejected based on a e b i ~ u i t y (N): Rejected based on gc_conLent: Rejected based on p r i c e r Te: ReJected based on s e l f - a n n e a l i n ~ Rejected based on oLher-annealin~: Nunber accepted: Antisense: T o t a l considered: Rejected based on a n b i g u i L y (N): Rejected based on ¢c_content: Rejected based on p r i n e r Tn: ANNEALINi Rejected based on s e l f - a n n e a l i n e : Annealing Sco, Rejected based on other-annealing: Prlner-sel, Nunber accepted: PRODUCTS Priner-prin, T o t a l considered: Rejected based on i d e n t i c a l endphs: Priner-produ Rejected based on length: Priner-othe Rejected based on p r i m e r - p r i c e r annealine: Rejected based on priner-producL annealing: RT Scor, Rejected based on e e l t i n g LenperaEure: Rejected based on gc_conLenL: CG Scor, Rejected based on diFFerence in Tn: gt Anbig Nunber accepted:
PRIMER CONSTRAINTS
Product l e n e t h ( b p ) :
248
434
309 G3 121 52 2
0 31 594 20 405 119 11 0
35 1085 G34 0 124 0
0 0
0 327
.... CANCEL
i[INPUT CONSTRAINTS EROH FILE: JIS"VE CONSTRAINTS TO FILE: ]~180oSeqodet"
FIGURE 1 Screen dump of OSP display. See text for explanation.
graphical display version of OSP has also been developed, which runs under X windows (Fig. 1). Clicking on the O H I O N S box brings up a m e n u with the program options. The sequence(s) are entered either by specifying a file name(s) or by typing or "pasting" sequence into a sequence window. Any constraint or weight may be altered by entering values in a "constraints" or "weights" text window. The user m a y alter the default values displayed in the constraints window by specifying a "constraint file" n a m e on the comm a n d line. Analysis is initiated by clicking on the ANALYZE box. The graphic results w i n d o w displays a line representing 126 PCR Methods and Applications
the input sequence(s), with locations of the ranked primers displayed beneath it. Clicking on a primer causes information concerning it to be displayed in the text results window. The n u m b e r of pairs accepted, along with a breakdown by constraint of the n u m ber rejected, can be displayed by clicking on OLIGO INFO. Any constraint can be changed and the program rerun by clicking on the appropriate buttons. Clicking on the SAVE box outputs a specified n u m b e r of primer pairs to a file. If the sequence file is accompanied by a trace data file from a fluorescent sequencing m a c h i n e (ABI 373A or Pharmacia A.L.F.), clicking above the
line representing the sequence calls up the display of the trace (using the ted trace editor ¢7)) for that portion of the sequence. This feature has proven useful for verifying accuracy of the sequence in the vicinity of candidate primers, in the C. elegans sequencing project. RESULTS
OSP has been used to choose over 365 sequencing primers and over 300 PCR primer pairs. Table 2 gives the "success rates" (proportion of primers successfully used in sequencing reactions) for four cosmids in the C. elegans g e n o m e sequencing project. (8) This project involves a two-stage strategy for sequenc-
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
TABLE 2 0 S P - s e l e c t e d C. elegans S e q u e n c i n g Primer Success Rates
Project(a)
Template DNA
B0303 ZK370 ZK643 F59B2
plasmid plasmid plasmid/M13 plasmid/M13
Number successful/ total(%) b
Data source
95/102 (93.1) 44/45 (97.8) 109/1 lO (99.1) 98/100 (98.0)
R. Wilson R. Wilson J. Sulston T. Hawkins
aCosmids from C. elegans physical map. OSP default constraint settings were used, with the following exceptions: rain. primer Tm = SO, max. -- 60; for ZK643 and F59B2, primer length constraints were min. = 17, max. = 23, and rain. primer G+C content = 30%. For sequencing reaction conditions, see ref. 9. bFor 20 of the ZK643 and F59B2 primers, it was necessary, to rerun the reactions at a higher temperature to obtain specific priming; these are included among the successes.
ing cosmids from the C. elegans physical map: an initial "shotgun" stage, in w h i c h a single sequence tract is generated from each of several h u n d r e d largeinsert M13 or p l a s m i d subclones of the c o s m i d a n d assembled i n t o contigs, followed by a "walking" stage to fill gaps b e t w e e n contigs, to o b t a i n d o u b l e - s t r a n d e d coverage in all regions, a n d to resolve a n y r e m a i n i n g seq u e n c e ambiguities. OSP is used to c h o o s e the w a l k i n g primers. The success rates for PCR primers are given in Table 3. The bulk of these primers were selected for the C. elegans physical m a p p i n g project, a n d for t h e 7 and X chromosome mapping projects in t h e W a s h i n g t o n University G e n o m e Center. For t h e latter (in w h i c h sequences derived from )~ or M13 g e n o m i c clones were used to c h o o s e t h e primers) we i n c l u d e as "successes" all cases in w h i c h a b a n d of t h e a n t i c i p a t e d size was o b t a i n e d from
a m p l i f i c a t i o n of the g e n o m i c DNA source from w h i c h t h e clone library was prepared, w h e t h e r or n o t the seq u e n c e u l t i m a t e l y t u r n e d o u t to be single c o p y in t h e g e n o m e or m a p p e d to the desired h u m a n c h r o m o s o m e . The success rates in these tables are likely to be u n d e r e s t i m a t e s of OSP's effectiveness at c h o o s i n g primers, since failures m a y reflect use of incorrect sequence, o l i g o n u c l e o t i d e synthesis errors, reaction failures, or (in t h e g e n o m i c PCR cases) s e q u e n c i n g from clones w h i c h m a y n o t be faithful replicas of t h e g e n o m e , rather t h a n p o o r choices by OSP. In particular, c l o n i n g artifacts a l m o s t c e r t a i n l y acc o u n t for a substantial fraction of the PCR failures. In t h e c h r o m o s o m e 7 project, m o s t of the failed primer pairs tested were f o u n d to a m p l i f y the clone DNA successfully, e v e n w h e n it was m i x e d w i t h g e n o m i c DNA such t h a t t h e c l o n e sequence was present at t h e
TABLE 30SP-selected PCR Primer Pair Success Rates
Project
Template DNA
Chromosome 7a
human genomic
Chromosome 13
human genomic
Chromosome X C. elegans physical map C. elegans physical map
human genomic C. elegans cosmid C. elegans YAC
Number successful/ total(%)
Data source
63/80 (78.8) b SS/6S (84.6) c 14/17 (82.4) b 24/24 (100.0) d 19/29 (65.5) b 46/46 (100.0) 41/44 (93.2)
E.D. Green E.D. Green P. Kwok P. Kwok J. Kere Y. Kozono Y. Kozono
aDefault OSP constraint settings were relaxed as follows: min. product Length, 60; max. product Tm, 82; max. G+C content, 60%; max. difference between primer Tin's, 3. Sequences for which OSP found no primer pairs meeting all constraints were skipped. See ref. 10 for a description of the PCR conditions used. bSequences derived from M13 clones of flow-sorted human chromosomes. CSequences derived from "bubble PCR" of ~. clones. dSequences derived from bubble PCR of YAC ends. Many of these primers were also used for sequencing (successfully, in all cases).
same c o n c e n t r a t i o n as single-copy g e n o m i c sequences (E.D. Green, pers. c o m m . ) . Thus, these failures m o r e likely result from a fraction of t h e clones t h a t c o n t a i n rearranged or foreign DNA, rather t h a n from n o n r o b u s t primers. In this light, it is i n t e r e s t i n g t h a t the PCR success rates appear to d e p e n d significantly o n the m e t h o d used to c o n s t r u c t t h e clones or subclones from w h i c h t h e sequence was d e t e r m i n e d : t h e overall success rate was o n l y 76.2% for sequences inferred from M13 subclones of flow-sorted hum a n c h r o m o s o m e s (lines 1, 3, a n d 5 in Table 3), while for sequences o b t a i n e d by o t h e r m e t h o d s (lines 2 a n d 4 in Table 3), it was 88.8%. This suggests t h a t , at least in these e x p e r i m e n t s , M13 c l o n i n g f r o m small a m o u n t s of source DNA m a y h a v e been m o r e p r o n e to r e a r r a n g e m e n t s or i n c l u s i o n of c o n t a m i n a t i n g DNA, a n d t h a t OSP's success rate at c h o o s i n g h u m a n g e n o m i c PCR primers m a y a p p r o a c h 90% w h e n the g e n o m i c sequence is accurately k n o w n . The v a r i a t i o n in success rates a m o n g t h e m a p p i n g projects p r o b a b l y also reflects differing efforts to optimize t h e PCR c o n d i t i o n s ; for example, a larger variety of t e m p e r a t u r e regimes a n d buffers were tried in t h e c h r o m o s o m e 7 project (cf. ref. 10) t h a n in the X c h r o m o s o m e project, w h i c h m a y a c c o u n t for its h i g h e r success rate. It s h o u l d also be n o t e d t h a t these success rates will likely be i m p r o v e d by OSP's testing for base-pairing w i t h seq u e n c e s in the "other sequence" file, w h i c h was n o t available at t h e t i m e these e x p e r i m e n t s were d o n e . DISCUSSION The detailed kinetics of PCR a n d sequencing reactions, in particular p r i m e r - t e m p l a t e interactions, are n o t well u n d e r s t o o d , a n d as a result the criteria c u r r e n t l y used to c h o o s e primers are largely empirical. Primer T m is of o b v i o u s relevance for t h e t e m p e r a t u r e cycling protocols. Primer %G+C c o n t e n t is also t h o u g h t to be i m p o r t a n t , a l t h o u g h it is u n c l e a r w h e t h e r this is because of its i n f l u e n c e o n s e c o n d a r y structure or n o n s p e c i f i c a n n e a l i n g to t e m p l a t e DNA. Specificity of t h e p r i m i n g r e a c t i o n s h o u l d be enh a n c e d , a n d p r i m e r s e c o n d a r y structure a n d p r i m e r - p r i m e r i n t e r a c t i o n s
PCR Methods and Applications 127
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
largely avoided, by minimizing potential primer base-pairing with nontarget sequences in the reaction mixture, particularly those present in high copy number. Thus, it is desirable to ensure low annealing of the primer with itself; with the other primer (in PCR), with (nontarget) sites in the amplified product; with repetitive elements, w h e n the template is genomic DNA; and additionally, w h e n the template is cloned DNA, with any nontarget sequences k n o w n to be present in the clone (such as the vector sequence). OSP allows the calculation of a simple base-pairing or "annealing" score for the primer with each of these types of sequences. In comparison to other primer selection programs we have examined, OSP is unusual in the range of usersettable parameters employed (for example, base-pairing involving the internal primer sequence can be scored differently from base-pairing at the 3 ' end, w h i c h is presumably more likely t o lead to nonspecific priming), in allowing prescreening against other sequences, and in the flexibility allowed the user in setting constraints and ranking candidate primers. Any criterion can be altered to find primerpairs in a particularly difficult sequence or to meet different experimental conditions. This flexibility allows the user's selection criteria to evolve in the light of laboratory experience. As yet, no m e t h o d for choosing primers has been tested in a controlled experiment, and OSP is no exception. Since the criteria it uses are m u c h the same as those used by investigators without such a program available to them, its success rates quite possibly do not exceed those attainable by an expert without computer assistance. Nonetheless, at a m i n i m u m OSP allows the choice to be made rapidly and automatically, and has success rates that exceed those often reported anecdotally. AVAILABILITY
C language source code for OSP is available (for research purposes only) at no cost from the authors, in either the text output version (tested for VAX/VMS, PC, MAC, a n d SUN Sparcstations), or interactive X windows graphics version (tested for SUN Sparcstations).
128 PCR Methods and Applications
ACKNOWLEDGMENTS
We t h a n k Pui Kwok, J o h n Sulston, Eric Green, Trevor Hawkins, J u h a Kere, Bob Waterston, Rick Wilson, and Yuko Kozono for suggestions to improve the program's ease of use, and for providing data concerning their primer success rates. This work was supported by National Institutes of Health grants PS0-HG00201 and R01-HG00136, and by a New Faculty Award from the Lucille P. Markey Charitable Trust. REFERENCES
1. Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. 74: 5463-5467. 2. Mullis, K.B. and F.A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155: 335-350. 3. Lowe, T., J. Sharefkin, S.Q. Yang, and C.W. Dieffenbach. 1990. A computer program for selection of oligonucleotide primers for polymerase chain reactions. Nucleic Acids Res. 18: 1757-1761. 4. Rychlik, W. and R.E. Rhoads. 1989. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing, and in vitro amplification of DNA. Nucleic Acids Res. 17: 8543-8551. 5. Olson, M., L. Hood, C. Cantor, and D. Botstein. 1989. A common language for physical mapping of the human genome. Science 245: 1434-1435. 6. Bolton, E.T. and B.J. McCarthy. 1962. A general method for the isolation of RNA complementary to DNA. Proc. Natl. Acad. Sci. 48: 1390-1394. 7. Gleeson, T. and L. Hillier, in preparation. 8. Sulston, J., R. Ainscough, M. Berks, M. Craxton, A. Coulson, S. Dear, Z. Du, R. Durbin, P. Green, N. Halloran, T. Hawkins, L. Hillier, C. Huynh, Y. Kozono, C. Lee, B. Lutterbach, M. Metzstein, Q. Qiu, R. Shownkeen, R. Staden, J. Thierry-Mieg, K. Thomas, R. Wilson, and R. Waterston, in preparation. 9. Hawkins, T.L. and J.E. Sulston. 1990. Automated fluorescent primer walking. Techniques 2:307-310. 10. Green, E.D., R.M. Mohr, J.R. Idol, M. Jones, J.M. Buckingham, L.L. Deaven, R.K. Moyzis, and M.V. Olson. 1991.
Systematic generation of sequencetagged sites (STSs) for physical mapping of human chromosomes: Application to the mapping of human chromosome 7 using yeast artificial chromosomes. Genomics (in press). Received August 1, 1991; accepted September 17, 1991.
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
OSP: a computer program for choosing PCR and DNA sequencing primers. L Hillier and P Green Genome Res. 1991 1: 124-128 Access the most recent version at doi:10.1101/gr.1.2.124
References
This article cites 7 articles, 5 of which can be accessed free at: http://genome.cshlp.org/content/1/2/124.full.html#ref-list-1
Creative Commons License
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.
Email Alerting Service
Receive free email alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here.
To subscribe to Genome Research go to:
http://genome.cshlp.org/subscriptions
Copyright © Cold Spring Harbor Laboratory Press
OSP: A Computer Program for Choosing PCR and DNA
Sequencing Primers LaDeana Hillier and Philip Green Genetics Department, Washington University School of Medicine St. Louis, Missouri 63110
OSP (Oligonucleotide Selection Program) selects ollgonucleotide primers for DNA sequencing 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, and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary 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 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 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 meeting all constraints are ranked by a "combined score," a userdefinable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the Caenorhabditis elegons genome sequencing project and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 9 6 % 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. 124 PCR Methods and Applications
DNA sequencing by the Sanger chain-termination method, (1) and the polymerase chain reaction (PCR), (2) are ubiquitous molecular biology tools that require oligonucleotides for priming DNA synthesis. Not all primers work, and those that do vary widely in their resistance to changes in reaction conditions, a situation that has spurred the development of several computer programs for choosing primers. (3,4) The onset of major mapping and sequencing efforts as part of the Genome Initiative has increased the demand for efficient primer selection. OSP (Oligonucleotide Selection Program) was designed to satisfy the needs of two projects: the Washington University Genome Center's project to map human chromosomes 7 and X, which will require PCR assays to detect well over 1000 sequence-tagged sites (STSs) (s) in h u m a n genomic DNA, and
the Caenorhabditis elegans genome sequencing project, which is presently using a mixed shotgun sequencing and walking strategy requiring selection of sequencing primers (roughly 100 per cosmid) for the walking steps. Major requirements for OSP were ease of use and flexibility in setting primer selection criteria, so that the program could evolve in the light of experience gained in both projects. In this paper we describe O S P , and summarize results obtained using it.
METHODS OSP: Program Design and Operation Input of sequences For selecting PCR primers, the user provides either one sequence that includes the region to be amplified or two sequences flanking that region; for selec-
ting sequencing primers, a single sequence must be provided. Either strand may be given, and ambiguous nucleotides within the sequence are permitted (although primers containing ambiguous nucleotides are rejected). Sequences may either be entered at run time (in the graphics version of the program, they may be "pasted" in from another window) or provided as a text file(s) in a variety of possible formats. The user may confine the search for candidate primers to particular regions of the input sequence. The primer locations may also be specified exactly, if the user wishes to compute scores or other characteristics for previously selected primers.
Constraints Primer and amplified product characteristics that may be constrained are listed in Table 1, along with OSP's default values, which have been found to work well in several mapping and sequencing projects (see Results). The constraint criteria are those thought (largely on the basis of anecdotal evidence) to be important to ensure sensitive and specific priming: for example, if G+C content is too high, the primer may be more prone to adopt secondary structure or to anneal nonspecifically to GC-rich regions of the template DNA, while if it is too low the primer may not anneal to its target sequence. If the user does not wish to use the defaults, modified constraint values for any or all parameters may be provided in a "constraint file," and/or entered directly at run time (the constraints for any parameter can also be disabled). "Annealing scores" are intended to provide simple measures of basepairing propensity of the primer with
1:124-128@1991 by Cold Spring Harbor Laboratory PressISSN1054-9803/91 $3.00
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
TABLE 1 User-constrainable Parameters, Default Limits, a n d Default Weights
Minimum
Maximum
Weight
Single primer parameters 3 ' nucleotidesa Primer length (nucleotides)c Primer G+C content (%) Primer Tm (Celsius) d Annealing scorese Primer-self internal Primer-self 3' Primer-other internal Primer-other 3 '
Sa
18 40.0 50.5 --
mb
22 55.0 55.0
0.0 0.0 0.0
14.0
1.0
--
8.0 NSf
2.0 0.0
--
NS
0.0
--
Primer pair parameters Product length (bp)g Product G÷C content (%) Product Tm (Celsius) Primer Tm difference (Celsius) Annealing scores Primer-primer internal Primer-primer 3 ' Primer-product internal Primer-product 3'
100 40.0 70.0 --
D
300 $5.0 90.0 2.0
0.0 0.0 0.0 0.0
14.0 8.0 NS NS
1.0 2.0 0.0 0.0
aAny string of 3 ' terminal nucleotide(s) for the primers may be specified, with all standard ambiguity codes allowed. The default is a single nucleotide S, which matches either C or G. b(--)Not meaningful. CValues shown are for PCR primers. For sequencing primers, defaults are rain. = 17, max. = 18. dTm (melting temperature) is calculated as: 62.3 + 0.41(%(G+C)) - 500/length. (6) Any ambiguous nucleotides are ignored. eSee text for explanation. f(NS) Not set (in OSP defaults). gFor sequencing primers, product length is the distance from the end of the sequence. Default constraints are min. = 40, max. = 100. the various n o n t a r g e t sequences present in the reaction. They are c o m p u t e d as follows: The user may define a score for an A/T base pair (default value, 2) and for a C/G base pair (default value, 4). For a n y a l i g n m e n t of two sequences (with the first sequence written 5 ' t o 3 ' , and the second one 3 ' t o 5 ' ) scores are s u m m e d over contiguous c o m p l e m e n t a r y nucleotide pairs; the m a x i m u m such score, taken over all contiguous blocks in all alignments, is the internal a n n e a l i n g score. The 3 ' a n n e a l i n g score (which is c o m p u t e d and weighted separately because it likely has a greater influence on nonspecific priming) is based only o n contiguous c o m p l e m e n t a r y matches that include the 3 ' e n d of the primer. Annealing scores are calculated for the primer with itself, with the other primer, with the amplified product, a n d with a user supplied set of "other" sequences. At a m i n i m u m , the latter would typically include repetitive sequences w h e n the template is g e n o m i c
DNA, or vector sequence w h e n the template is cloned DNA. It could also include other primer sequences if one is interested in "multiplexing" multiple PCR primer pairs in the same reaction tube. For example, the a l i g n m e n t 5 ' -ACGGATTGTGCCGTATTG-3 ' 3' TGACACGTGTAGACGAGG-5 ' of two candidate primers would yield an internal a n n e a l i n g score of 16 (using the default base-pair scores), corresponding to the pairing of TGTGC on the top strand with ACACG o n the bottom, and a 3 ' a n n e a l i n g score of 6, corresponding to the pairing of TG at the 3 ' e n d of the first primer with AC. Since the default internal a n n e a l i n g cutoff (14.0) is exceeded, this pair would be rejected by OSP.
position w i t h i n the sequence, it tests candidate primers h a v i n g that position as the 3 ' terminus, e x a m i n i n g in order the criteria in the upper half of Table 1. Primers c o n t a i n i n g an ambiguous nucleotide are automatically rejected. Primers with the same 3 ' e n d are considered in order of increasing length; this allows consideration of longer primers to be aborted w h e n " m o n o t o n ically increasing" parameters (e.g., primer-self or primer-other annealing) exceed their constraint values. If all criteria are satisfied, the primer is saved in a list of candidates for that strand.
PCR Primer Pair Selection Algorithm Following construction of lists of candidate primers for each strand, candidate primer pairs (one from each list) are considered. If several pairs have the same 3 ' ends for b o t h primers, at most one pair (the shortest) is accepted. For each candidate pair, constraints involving the amplified product and primer pair (lower half of Table 1) are tested, and any pair meeting each applicable constraint is added to the list of valid pairs. Once all possible primer pairs have been considered, t h e y are ranked based on their "combined score," w h i c h is a user-definable weighted sum of a n y of the parameters in Table 1. The default c o m b i n e d score (which uses the default weights in Table 1) is based o n the primer-self and primer-primer annealing scores, a n d weighs 3 ' annealing twice as m u c h as internal annealing. The user m a y supply different weights in the constraint file.
Program Output OSP notifies the user of the n u m b e r of primers (or primer pairs) accepted, a n d provides a table listing the n u m b e r of pairs rejected for each constraint. (This is often useful in cases where n o pair meets all constraints.) The user can t h e n choose to save (to a file) a ranked list of as m a n y of the primers as desired, along with their characteristics. The o u t p u t file also records the constraint values used and the n u m bers of primers or pairs accepted and rejected.
Primer Selection Algorithm OSP scans each strand in a 5 ' t o 3 ' direction for possible primers. For each
Graphic Interface A
menu-driven
"point
and
click"
PCR Methods and Applications 125
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
~lick
TYPE/PASTE IN SEQUENCE uiLh the 1 e l l eouse button ON any primer to have score i n f e r e a t i o n displayed here
217
PRODUCT CONSTRAINTS Min
G.c Conte.t(~):
.a. 130~ .in L4~ Na, [5t
) ]
min
1
Tn(Celsius):
.a.
~IC.NCEL
[10~
170. ~ [90.0
]
Length(bases):
Min
118
]
Ha.
12~
I
Min
140
]
.a.
155.
[
Min
150. ~
J
.a.
155.~
I
Difference in Te cuLoFF:
I 2.0
l
3" NucleoLides (S=G/C,g=A/T):
IS~
I
l G+C ConLent(X);
Tm(Celslus):
J
279
310
341
372
403
)RIMERS (Nunber o£ S-type sequences Found uas 183) Sense: T o t a l considered: Rejected based on a e b i ~ u i t y (N): Rejected based on gc_conLent: Rejected based on p r i c e r Te: ReJected based on s e l f - a n n e a l i n ~ Rejected based on oLher-annealin~: Nunber accepted: Antisense: T o t a l considered: Rejected based on a n b i g u i L y (N): Rejected based on ¢c_content: Rejected based on p r i n e r Tn: ANNEALINi Rejected based on s e l f - a n n e a l i n e : Annealing Sco, Rejected based on other-annealing: Prlner-sel, Nunber accepted: PRODUCTS Priner-prin, T o t a l considered: Rejected based on i d e n t i c a l endphs: Priner-produ Rejected based on length: Priner-othe Rejected based on p r i m e r - p r i c e r annealine: Rejected based on priner-producL annealing: RT Scor, Rejected based on e e l t i n g LenperaEure: Rejected based on gc_conLenL: CG Scor, Rejected based on diFFerence in Tn: gt Anbig Nunber accepted:
PRIMER CONSTRAINTS
Product l e n e t h ( b p ) :
248
434
309 G3 121 52 2
0 31 594 20 405 119 11 0
35 1085 G34 0 124 0
0 0
0 327
.... CANCEL
i[INPUT CONSTRAINTS EROH FILE: JIS"VE CONSTRAINTS TO FILE: ]~180oSeqodet"
FIGURE 1 Screen dump of OSP display. See text for explanation.
graphical display version of OSP has also been developed, which runs under X windows (Fig. 1). Clicking on the O H I O N S box brings up a m e n u with the program options. The sequence(s) are entered either by specifying a file name(s) or by typing or "pasting" sequence into a sequence window. Any constraint or weight may be altered by entering values in a "constraints" or "weights" text window. The user m a y alter the default values displayed in the constraints window by specifying a "constraint file" n a m e on the comm a n d line. Analysis is initiated by clicking on the ANALYZE box. The graphic results w i n d o w displays a line representing 126 PCR Methods and Applications
the input sequence(s), with locations of the ranked primers displayed beneath it. Clicking on a primer causes information concerning it to be displayed in the text results window. The n u m b e r of pairs accepted, along with a breakdown by constraint of the n u m ber rejected, can be displayed by clicking on OLIGO INFO. Any constraint can be changed and the program rerun by clicking on the appropriate buttons. Clicking on the SAVE box outputs a specified n u m b e r of primer pairs to a file. If the sequence file is accompanied by a trace data file from a fluorescent sequencing m a c h i n e (ABI 373A or Pharmacia A.L.F.), clicking above the
line representing the sequence calls up the display of the trace (using the ted trace editor ¢7)) for that portion of the sequence. This feature has proven useful for verifying accuracy of the sequence in the vicinity of candidate primers, in the C. elegans sequencing project. RESULTS
OSP has been used to choose over 365 sequencing primers and over 300 PCR primer pairs. Table 2 gives the "success rates" (proportion of primers successfully used in sequencing reactions) for four cosmids in the C. elegans g e n o m e sequencing project. (8) This project involves a two-stage strategy for sequenc-
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
TABLE 2 0 S P - s e l e c t e d C. elegans S e q u e n c i n g Primer Success Rates
Project(a)
Template DNA
B0303 ZK370 ZK643 F59B2
plasmid plasmid plasmid/M13 plasmid/M13
Number successful/ total(%) b
Data source
95/102 (93.1) 44/45 (97.8) 109/1 lO (99.1) 98/100 (98.0)
R. Wilson R. Wilson J. Sulston T. Hawkins
aCosmids from C. elegans physical map. OSP default constraint settings were used, with the following exceptions: rain. primer Tm = SO, max. -- 60; for ZK643 and F59B2, primer length constraints were min. = 17, max. = 23, and rain. primer G+C content = 30%. For sequencing reaction conditions, see ref. 9. bFor 20 of the ZK643 and F59B2 primers, it was necessary, to rerun the reactions at a higher temperature to obtain specific priming; these are included among the successes.
ing cosmids from the C. elegans physical map: an initial "shotgun" stage, in w h i c h a single sequence tract is generated from each of several h u n d r e d largeinsert M13 or p l a s m i d subclones of the c o s m i d a n d assembled i n t o contigs, followed by a "walking" stage to fill gaps b e t w e e n contigs, to o b t a i n d o u b l e - s t r a n d e d coverage in all regions, a n d to resolve a n y r e m a i n i n g seq u e n c e ambiguities. OSP is used to c h o o s e the w a l k i n g primers. The success rates for PCR primers are given in Table 3. The bulk of these primers were selected for the C. elegans physical m a p p i n g project, a n d for t h e 7 and X chromosome mapping projects in t h e W a s h i n g t o n University G e n o m e Center. For t h e latter (in w h i c h sequences derived from )~ or M13 g e n o m i c clones were used to c h o o s e t h e primers) we i n c l u d e as "successes" all cases in w h i c h a b a n d of t h e a n t i c i p a t e d size was o b t a i n e d from
a m p l i f i c a t i o n of the g e n o m i c DNA source from w h i c h t h e clone library was prepared, w h e t h e r or n o t the seq u e n c e u l t i m a t e l y t u r n e d o u t to be single c o p y in t h e g e n o m e or m a p p e d to the desired h u m a n c h r o m o s o m e . The success rates in these tables are likely to be u n d e r e s t i m a t e s of OSP's effectiveness at c h o o s i n g primers, since failures m a y reflect use of incorrect sequence, o l i g o n u c l e o t i d e synthesis errors, reaction failures, or (in t h e g e n o m i c PCR cases) s e q u e n c i n g from clones w h i c h m a y n o t be faithful replicas of t h e g e n o m e , rather t h a n p o o r choices by OSP. In particular, c l o n i n g artifacts a l m o s t c e r t a i n l y acc o u n t for a substantial fraction of the PCR failures. In t h e c h r o m o s o m e 7 project, m o s t of the failed primer pairs tested were f o u n d to a m p l i f y the clone DNA successfully, e v e n w h e n it was m i x e d w i t h g e n o m i c DNA such t h a t t h e c l o n e sequence was present at t h e
TABLE 30SP-selected PCR Primer Pair Success Rates
Project
Template DNA
Chromosome 7a
human genomic
Chromosome 13
human genomic
Chromosome X C. elegans physical map C. elegans physical map
human genomic C. elegans cosmid C. elegans YAC
Number successful/ total(%)
Data source
63/80 (78.8) b SS/6S (84.6) c 14/17 (82.4) b 24/24 (100.0) d 19/29 (65.5) b 46/46 (100.0) 41/44 (93.2)
E.D. Green E.D. Green P. Kwok P. Kwok J. Kere Y. Kozono Y. Kozono
aDefault OSP constraint settings were relaxed as follows: min. product Length, 60; max. product Tm, 82; max. G+C content, 60%; max. difference between primer Tin's, 3. Sequences for which OSP found no primer pairs meeting all constraints were skipped. See ref. 10 for a description of the PCR conditions used. bSequences derived from M13 clones of flow-sorted human chromosomes. CSequences derived from "bubble PCR" of ~. clones. dSequences derived from bubble PCR of YAC ends. Many of these primers were also used for sequencing (successfully, in all cases).
same c o n c e n t r a t i o n as single-copy g e n o m i c sequences (E.D. Green, pers. c o m m . ) . Thus, these failures m o r e likely result from a fraction of t h e clones t h a t c o n t a i n rearranged or foreign DNA, rather t h a n from n o n r o b u s t primers. In this light, it is i n t e r e s t i n g t h a t the PCR success rates appear to d e p e n d significantly o n the m e t h o d used to c o n s t r u c t t h e clones or subclones from w h i c h t h e sequence was d e t e r m i n e d : t h e overall success rate was o n l y 76.2% for sequences inferred from M13 subclones of flow-sorted hum a n c h r o m o s o m e s (lines 1, 3, a n d 5 in Table 3), while for sequences o b t a i n e d by o t h e r m e t h o d s (lines 2 a n d 4 in Table 3), it was 88.8%. This suggests t h a t , at least in these e x p e r i m e n t s , M13 c l o n i n g f r o m small a m o u n t s of source DNA m a y h a v e been m o r e p r o n e to r e a r r a n g e m e n t s or i n c l u s i o n of c o n t a m i n a t i n g DNA, a n d t h a t OSP's success rate at c h o o s i n g h u m a n g e n o m i c PCR primers m a y a p p r o a c h 90% w h e n the g e n o m i c sequence is accurately k n o w n . The v a r i a t i o n in success rates a m o n g t h e m a p p i n g projects p r o b a b l y also reflects differing efforts to optimize t h e PCR c o n d i t i o n s ; for example, a larger variety of t e m p e r a t u r e regimes a n d buffers were tried in t h e c h r o m o s o m e 7 project (cf. ref. 10) t h a n in the X c h r o m o s o m e project, w h i c h m a y a c c o u n t for its h i g h e r success rate. It s h o u l d also be n o t e d t h a t these success rates will likely be i m p r o v e d by OSP's testing for base-pairing w i t h seq u e n c e s in the "other sequence" file, w h i c h was n o t available at t h e t i m e these e x p e r i m e n t s were d o n e . DISCUSSION The detailed kinetics of PCR a n d sequencing reactions, in particular p r i m e r - t e m p l a t e interactions, are n o t well u n d e r s t o o d , a n d as a result the criteria c u r r e n t l y used to c h o o s e primers are largely empirical. Primer T m is of o b v i o u s relevance for t h e t e m p e r a t u r e cycling protocols. Primer %G+C c o n t e n t is also t h o u g h t to be i m p o r t a n t , a l t h o u g h it is u n c l e a r w h e t h e r this is because of its i n f l u e n c e o n s e c o n d a r y structure or n o n s p e c i f i c a n n e a l i n g to t e m p l a t e DNA. Specificity of t h e p r i m i n g r e a c t i o n s h o u l d be enh a n c e d , a n d p r i m e r s e c o n d a r y structure a n d p r i m e r - p r i m e r i n t e r a c t i o n s
PCR Methods and Applications 127
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
largely avoided, by minimizing potential primer base-pairing with nontarget sequences in the reaction mixture, particularly those present in high copy number. Thus, it is desirable to ensure low annealing of the primer with itself; with the other primer (in PCR), with (nontarget) sites in the amplified product; with repetitive elements, w h e n the template is genomic DNA; and additionally, w h e n the template is cloned DNA, with any nontarget sequences k n o w n to be present in the clone (such as the vector sequence). OSP allows the calculation of a simple base-pairing or "annealing" score for the primer with each of these types of sequences. In comparison to other primer selection programs we have examined, OSP is unusual in the range of usersettable parameters employed (for example, base-pairing involving the internal primer sequence can be scored differently from base-pairing at the 3 ' end, w h i c h is presumably more likely t o lead to nonspecific priming), in allowing prescreening against other sequences, and in the flexibility allowed the user in setting constraints and ranking candidate primers. Any criterion can be altered to find primerpairs in a particularly difficult sequence or to meet different experimental conditions. This flexibility allows the user's selection criteria to evolve in the light of laboratory experience. As yet, no m e t h o d for choosing primers has been tested in a controlled experiment, and OSP is no exception. Since the criteria it uses are m u c h the same as those used by investigators without such a program available to them, its success rates quite possibly do not exceed those attainable by an expert without computer assistance. Nonetheless, at a m i n i m u m OSP allows the choice to be made rapidly and automatically, and has success rates that exceed those often reported anecdotally. AVAILABILITY
C language source code for OSP is available (for research purposes only) at no cost from the authors, in either the text output version (tested for VAX/VMS, PC, MAC, a n d SUN Sparcstations), or interactive X windows graphics version (tested for SUN Sparcstations).
128 PCR Methods and Applications
ACKNOWLEDGMENTS
We t h a n k Pui Kwok, J o h n Sulston, Eric Green, Trevor Hawkins, J u h a Kere, Bob Waterston, Rick Wilson, and Yuko Kozono for suggestions to improve the program's ease of use, and for providing data concerning their primer success rates. This work was supported by National Institutes of Health grants PS0-HG00201 and R01-HG00136, and by a New Faculty Award from the Lucille P. Markey Charitable Trust. REFERENCES
1. Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. 74: 5463-5467. 2. Mullis, K.B. and F.A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155: 335-350. 3. Lowe, T., J. Sharefkin, S.Q. Yang, and C.W. Dieffenbach. 1990. A computer program for selection of oligonucleotide primers for polymerase chain reactions. Nucleic Acids Res. 18: 1757-1761. 4. Rychlik, W. and R.E. Rhoads. 1989. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing, and in vitro amplification of DNA. Nucleic Acids Res. 17: 8543-8551. 5. Olson, M., L. Hood, C. Cantor, and D. Botstein. 1989. A common language for physical mapping of the human genome. Science 245: 1434-1435. 6. Bolton, E.T. and B.J. McCarthy. 1962. A general method for the isolation of RNA complementary to DNA. Proc. Natl. Acad. Sci. 48: 1390-1394. 7. Gleeson, T. and L. Hillier, in preparation. 8. Sulston, J., R. Ainscough, M. Berks, M. Craxton, A. Coulson, S. Dear, Z. Du, R. Durbin, P. Green, N. Halloran, T. Hawkins, L. Hillier, C. Huynh, Y. Kozono, C. Lee, B. Lutterbach, M. Metzstein, Q. Qiu, R. Shownkeen, R. Staden, J. Thierry-Mieg, K. Thomas, R. Wilson, and R. Waterston, in preparation. 9. Hawkins, T.L. and J.E. Sulston. 1990. Automated fluorescent primer walking. Techniques 2:307-310. 10. Green, E.D., R.M. Mohr, J.R. Idol, M. Jones, J.M. Buckingham, L.L. Deaven, R.K. Moyzis, and M.V. Olson. 1991.
Systematic generation of sequencetagged sites (STSs) for physical mapping of human chromosomes: Application to the mapping of human chromosome 7 using yeast artificial chromosomes. Genomics (in press). Received August 1, 1991; accepted September 17, 1991.
Downloaded from genome.cshlp.org on May 23, 2015 - Published by Cold Spring Harbor Laboratory Press
OSP: a computer program for choosing PCR and DNA sequencing primers. L Hillier and P Green Genome Res. 1991 1: 124-128 Access the most recent version at doi:10.1101/gr.1.2.124
References
This article cites 7 articles, 5 of which can be accessed free at: http://genome.cshlp.org/content/1/2/124.full.html#ref-list-1
Creative Commons License
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.
Email Alerting Service
Receive free email alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here.
To subscribe to Genome Research go to:
http://genome.cshlp.org/subscriptions
Copyright © Cold Spring Harbor Laboratory Press
