Design of multiplex PCR primers using heuristic algorithm for sequential deletion applications

UNLABELLED The sequential deletion method is commonly applied to locate the functional domain of a protein. Unfortunately, manually designing primers for multiplex polymerase chain reaction (PCR) is a labor-intensive task. In order to speed up the experimental procedure and to improve the efficiency of producing PCR products, this paper proposes a multiplex PCR primers (MPCRPs) designer to design multiple forward primers with a single 3'-UTR reverse primer for extracting various N-terminal truncated mutants to quickly locate the functional domain of a cDNA sequence. Several factors, including melting temperature, primer length, GC content, internal self-complement, cross-dimerization, terminal limitation, and specificity, are used as the criteria for designing primers. This study obtains a near-optimal solution of primer sets that can be placed in as few test tubes as possible for one multiplex PCR experiment. RESULTS Homo sapiens ribosomal protein L5, Homo sapiens xylosyltransferase I, and Bacteriophage T4 gene product 11 were used as test examples to verify efficacy of the proposed algorithm. In addition, the designed primers of Homo sapiens ribosomal protein L5 cDNA were applied in multiplex PCR experiments. A total of 48 forward primers and one reverse primer were designed and used to duplicate N-terminal truncated mutants of different lengths from the protein. The primers were classified into eight tube groups (i.e., test tubes) held within the same temperature range (53-57 degrees C), and the validity of the PCR products were verified using polyacrylamide gel electrophoresis (PAGE) with the functional domain correctly located. A software implementation of the proposed algorithm useful in assisting the researcher to design primers for multiplex PCR experiments was developed and available upon request.

[1]  N. Templeton.  The Polymerase Chain Reaction History Methods, and Applications , 1992, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[2]  V. Mizrahi,et al.  Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy. , 2004, Tuberculosis.

[3]  A. Blennow,et al.  Functional domain organization of the potato alpha-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants. , 2005, The Biochemical journal.

[4]  M Vingron,et al.  Primer design for large scale sequencing. , 1998, Nucleic acids research.

[5]  Thomas Kämpke,et al.  Efficient primer design algorithms , 2001, Bioinform..

[6]  Ren-Hao Pan,et al.  NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants , 1996, Environmental health perspectives.

[7]  J. SantaLucia,et al.  A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Shaffer,et al.  Hybridization of synthetic oligodeoxyribonucleotides to ΦX 174 DNA: the effect of single base pair mismatch , 1979 .

[9]  S. Casjens,et al.  Molecular genetics of bacteriophage P22 scaffolding protein's functional domains. , 2005, Journal of molecular biology.

[10]  H. Blöcker,et al.  Predicting DNA duplex stability from the base sequence. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Elong Lin,et al.  The participation of 5 S rRNA in the co-translational formation of a eukaryotic 5 S ribonucleoprotein complex , 2001 .

[12]  Rung Ching Chen,et al.  NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants , 2007, Nucleic Acids Res..

[13]  Milton H Saier,et al.  Domain analysis of transcriptional regulators bearing PTS regulatory domains. , 2002, Research in microbiology.

[14]  Jain-Shing Wu,et al.  Primer design using genetic algorithm , 2004, Bioinform..

[15]  H. Seliger,et al.  PCR protocols — A guide to methods and applications , 1990 .

[16]  R A Gibbs,et al.  Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. , 1988, Nucleic acids research.

[17]  K. Itakura,et al.  Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. , 1979, Nucleic acids research.

[18]  C. Dieffenbach,et al.  A computer program for selection of oligonucleotide primers for polymerase chain reactions. , 1990, Nucleic acids research.

[19]  C. Levenson,et al.  Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. , 1990, Nucleic acids research.

[20]  C. Y. Lin,et al.  Primer Design Assistant (PDA): a web-based primer design tool , 2003, Nucleic Acids Res..

[21]  E Schütz,et al.  Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. , 2001, Clinical chemistry.

[22]  Simon Kasif,et al.  MuPlex: multi-objective multiplex PCR assay design , 2005, Nucleic Acids Res..

[23]  P. G. Leiman,et al.  Functional Role of the N-Terminal Domain of Bacteriophage T4-Gene Product 11 , 2005, Biochemistry (Moscow).

[24]  H R Garner,et al.  PRIMO: A primer design program that applies base quality statistics for automated large-scale DNA sequencing. , 1997, Genomics.

[25]  Joachim Kuhn,et al.  Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme. , 2006, The Biochemical journal.

[26]  Michael Young,et al.  Mutants of Mycobacterium tuberculosis Lacking Three of the Five rpf-Like Genes Are Defective for Growth In Vivo and for Resuscitation In Vitro , 2005, Infection and Immunity.