Cargando…

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

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 molecu...

Descripción completa

Detalles Bibliográficos
Autores principales: Mostaan, Saied, Ajorloo, Mehdi, Khanahmad, Hossein, Cohan, Reza Ahangari, Tehrani, Zahra Rikhtegaran, Rezaei, Maryam, Fazeli, Fateme, Behdani, Mehdi, Zare, Sakineh Karimi, Karimi, Zeinab, Mozhgani, Seyed Hamid Reza, Moukhah, Rasul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Medknow Publications & Media Pvt Ltd 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300601/
https://www.ncbi.nlm.nih.gov/pubmed/25625121
http://dx.doi.org/10.4103/2277-9175.148298
_version_ 1782353548674072576
author Mostaan, Saied
Ajorloo, Mehdi
Khanahmad, Hossein
Cohan, Reza Ahangari
Tehrani, Zahra Rikhtegaran
Rezaei, Maryam
Fazeli, Fateme
Behdani, Mehdi
Zare, Sakineh Karimi
Karimi, Zeinab
Mozhgani, Seyed Hamid Reza
Moukhah, Rasul
author_facet Mostaan, Saied
Ajorloo, Mehdi
Khanahmad, Hossein
Cohan, Reza Ahangari
Tehrani, Zahra Rikhtegaran
Rezaei, Maryam
Fazeli, Fateme
Behdani, Mehdi
Zare, Sakineh Karimi
Karimi, Zeinab
Mozhgani, Seyed Hamid Reza
Moukhah, Rasul
author_sort Mostaan, Saied
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4300601
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Medknow Publications & Media Pvt Ltd
record_format MEDLINE/PubMed
spelling pubmed-43006012015-01-26 A novel combined method for cost–benefit production of DNA ladders Mostaan, Saied Ajorloo, Mehdi Khanahmad, Hossein Cohan, Reza Ahangari Tehrani, Zahra Rikhtegaran Rezaei, Maryam Fazeli, Fateme Behdani, Mehdi Zare, Sakineh Karimi Karimi, Zeinab Mozhgani, Seyed Hamid Reza Moukhah, Rasul Adv Biomed Res Original Article 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. Medknow Publications & Media Pvt Ltd 2015-01-06 /pmc/articles/PMC4300601/ /pubmed/25625121 http://dx.doi.org/10.4103/2277-9175.148298 Text en Copyright: © 2015 Mostaan http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Original Article
Mostaan, Saied
Ajorloo, Mehdi
Khanahmad, Hossein
Cohan, Reza Ahangari
Tehrani, Zahra Rikhtegaran
Rezaei, Maryam
Fazeli, Fateme
Behdani, Mehdi
Zare, Sakineh Karimi
Karimi, Zeinab
Mozhgani, Seyed Hamid Reza
Moukhah, Rasul
A novel combined method for cost–benefit production of DNA ladders
title A novel combined method for cost–benefit production of DNA ladders
title_full A novel combined method for cost–benefit production of DNA ladders
title_fullStr A novel combined method for cost–benefit production of DNA ladders
title_full_unstemmed A novel combined method for cost–benefit production of DNA ladders
title_short A novel combined method for cost–benefit production of DNA ladders
title_sort novel combined method for cost–benefit production of dna ladders
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300601/
https://www.ncbi.nlm.nih.gov/pubmed/25625121
http://dx.doi.org/10.4103/2277-9175.148298
work_keys_str_mv AT mostaansaied anovelcombinedmethodforcostbenefitproductionofdnaladders
AT ajorloomehdi anovelcombinedmethodforcostbenefitproductionofdnaladders
AT khanahmadhossein anovelcombinedmethodforcostbenefitproductionofdnaladders
AT cohanrezaahangari anovelcombinedmethodforcostbenefitproductionofdnaladders
AT tehranizahrarikhtegaran anovelcombinedmethodforcostbenefitproductionofdnaladders
AT rezaeimaryam anovelcombinedmethodforcostbenefitproductionofdnaladders
AT fazelifateme anovelcombinedmethodforcostbenefitproductionofdnaladders
AT behdanimehdi anovelcombinedmethodforcostbenefitproductionofdnaladders
AT zaresakinehkarimi anovelcombinedmethodforcostbenefitproductionofdnaladders
AT karimizeinab anovelcombinedmethodforcostbenefitproductionofdnaladders
AT mozhganiseyedhamidreza anovelcombinedmethodforcostbenefitproductionofdnaladders
AT moukhahrasul anovelcombinedmethodforcostbenefitproductionofdnaladders
AT mostaansaied novelcombinedmethodforcostbenefitproductionofdnaladders
AT ajorloomehdi novelcombinedmethodforcostbenefitproductionofdnaladders
AT khanahmadhossein novelcombinedmethodforcostbenefitproductionofdnaladders
AT cohanrezaahangari novelcombinedmethodforcostbenefitproductionofdnaladders
AT tehranizahrarikhtegaran novelcombinedmethodforcostbenefitproductionofdnaladders
AT rezaeimaryam novelcombinedmethodforcostbenefitproductionofdnaladders
AT fazelifateme novelcombinedmethodforcostbenefitproductionofdnaladders
AT behdanimehdi novelcombinedmethodforcostbenefitproductionofdnaladders
AT zaresakinehkarimi novelcombinedmethodforcostbenefitproductionofdnaladders
AT karimizeinab novelcombinedmethodforcostbenefitproductionofdnaladders
AT mozhganiseyedhamidreza novelcombinedmethodforcostbenefitproductionofdnaladders
AT moukhahrasul novelcombinedmethodforcostbenefitproductionofdnaladders