Tehran University Medical Journal (May 2011)
Designing and constructing an 100 bp DNA Ladder by combining PCR and enzyme digestion methods
Abstract
"n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Molecular DNA markers are one of the most important tools in molecular biology labs. The size of DNA molecules is determined by comparing them with known bands of markers during gel electrophoresis. There are many different protocols to produce these kinds of molecular markers. In this study we have suggested an efficient strategy to produce molecular weight markers in industrial proportions."n"nMethods : To achieve the desired sizes of DNA fragments, a combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR), were used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the desired length of DNA fragments and produced the desired DNA fragment upon linearization. In the PCR method, the desired length of DNA fragments were cloned in multiple cloning sites of pTZ57R plasmid and in a PCR reaction, the new constructed plasmid was used as a template to produce the final fragment."n"nResults : Upon application of this strategy, 2000 and 3000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100 to 1500 bp fragments were produced during PCR using only a set of forward and reverse primers at the flanking region of pTZ57R multiple cloning site."n"nConclusion: The highest advantage of this cost-benefit approach is to produce different types of molecular weight markers by using an effective and short protocol."n"nKeywords: DNA markers, DNA Ladder, agarose gel electrophoresis, molecular weight.
