A novel combined method for cost-benefit production of DNA ladders

Authors

1 Department of R and D, Institute Pasteur of Iran, Tehran, Iran

2 Department of R and D, Institute Pasteur of Iran, Tehran; Department of Anatomical Sciences and Genetics, Isfahan Medicine School, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Virology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Background: Molecular deoxyribonucleic acid markers are one of the most important tools in molecular biology labs. The size of DNA molecule is determined by comparing them with known bands of markers during gel electrophoresis. In this study, we have suggested an efficient strategy to produce molecular weight markers in an industrial scale.
Materials and Methods: A combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR), was used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the bands of ladder and produce the DNA fragment by plasmid linearization through digestion. In the PCR method, the DNA fragments with length 102 bp lesser than the related bands in DNA ladder are amplified by PCR and cloned in pTZ57T/A cloning vector. Then, PCRs with forward and reverse 100-bp primers on the resulting plasmids amplify the ladder fragments. F100 and R100 primers bind to the backbone of pTZ57R (without insert) and amplify a 100-bp PCR product. PCR on the plasmid with insert amplifies DNA fragment with 102+ insert length bp size.
Results: Upon application of this strategy, 2000-10,000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100-1500 bp fragments were produced during PCR using only a set of forward and reverse (100 bp) primers.
Conclusion: The highest advantage of this cost-benefit approach is to produce different types of molecular weight markers by using an effective and short protocol.

Keywords

1.
Barvish Z, Davis C, Gitelman I. A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp. Electrophoresis 2007;28:900-2.  Back to cited text no. 1
    
2.
Hu A-Li W, Hartley JL, Heather JJ. Nucleic Acid Ladders. US Patent No. 0149640 A1, 2009.  Back to cited text no. 2
    
3.
Wang TY, Guo L, Zhang JH. Preparation of DNA ladder based on multiplex PCR technique. J Nucleic Acids 2010;2010:421803.  Back to cited text no. 3
    
4.
Dawson EP. Method for the multiplexed preparation of nucleic acid molecular weight markers and resultant products. US Patent No. 5,714,326, 1998.  Back to cited text no. 4
    
5.
Chang M, Wang JH, Lee HJ. Laboratory production of 100 base pair DNA molecular weight markers. J Biochem Biophys Methods 2008;70:1199-202.  Back to cited text no. 5
    
6.
Dongyi H, Longhai Z, Huazong Z, Ye C. Construction of DNA marker plasmids based on taq tailing activity and selective recovery of ligation products. Plant Mol Biol Rep 2008;26:316-23.  Back to cited text no. 6
    
7.
Holton TA, Graham MW. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res 1991;19:1156.  Back to cited text no. 7
    
8.
Saidijam M, Khanahmad H, Rikhtegaran Tehrani Z, Karimizare S, Shabab N, Behdani M. Designing and constructing an 100 bp DNA Ladder by combining PCR and enzyme digestion methods. Tehran Univ Med J 2011;69:75-82.  Back to cited text no. 8
    
9.
Hartley JL. Nucleic acid marker ladder for estimating mass. US Patent No. 7,132,520 B2, 2006.  Back to cited text no. 9
    
10.
Hartley JL, Gregori, Tamara J. Cloning multiple copies of a DNA segment. Gene 1981;13:347-53.  Back to cited text no. 10
    
11.
Hyman DE. DNA ladders. US Patent No. 5,840,575, 1998.  Back to cited text no. 11