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

Mostaan S, Ajorloo M, Khanahmad H, Cohan RA, Tehrani ZR, Rezaei M, Fazeli F, Behdani M, Zare SK, Karimi Z, Mozhgani SH, Moukhah R - Adv Biomed Res (2015)

Bottom Line: Then, PCRs with forward and reverse 100-bp primers on the resulting plasmids amplify the ladder fragments.Moreover, 100-1500 bp fragments were produced during PCR using only a set of forward and reverse (100 bp) primers.The highest advantage of this cost-benefit approach is to produce different types of molecular weight markers by using an effective and short protocol.

View Article: PubMed Central - PubMed

Affiliation: Department of R and D, Institute Pasteur of Iran, 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.

No MeSH data available.


Gel electrophoresis of purified fragments on 1% agarose gel: (a) 100-bp and (b) 1-kb DNA ladders. The standard DNA ladders were purchased from Fermentas Company
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Figure 1: Gel electrophoresis of purified fragments on 1% agarose gel: (a) 100-bp and (b) 1-kb DNA ladders. The standard DNA ladders were purchased from Fermentas Company

Mentions: In the enzymatic process, the five plasmids were successfully constructed based on pTZ57R which could be propagated in Top10 F` E. coli strain, because of its origin of replication and ampr gene. The other generated plasmids, pGFPIRES, pHH, and pUSHH, also had the same properties and could be amplified by the same system. All propagated plasmids were extracted from Top10 F`E. coli strain and subjected to enzymatic digestion by cheap restriction enzymes such as EcoRI, EcoRV, and KpnI to produce the desired size ladders [Figure 1a and b].


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

Mostaan S, Ajorloo M, Khanahmad H, Cohan RA, Tehrani ZR, Rezaei M, Fazeli F, Behdani M, Zare SK, Karimi Z, Mozhgani SH, Moukhah R - Adv Biomed Res (2015)

Gel electrophoresis of purified fragments on 1% agarose gel: (a) 100-bp and (b) 1-kb DNA ladders. The standard DNA ladders were purchased from Fermentas Company
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300601&req=5

Figure 1: Gel electrophoresis of purified fragments on 1% agarose gel: (a) 100-bp and (b) 1-kb DNA ladders. The standard DNA ladders were purchased from Fermentas Company
Mentions: In the enzymatic process, the five plasmids were successfully constructed based on pTZ57R which could be propagated in Top10 F` E. coli strain, because of its origin of replication and ampr gene. The other generated plasmids, pGFPIRES, pHH, and pUSHH, also had the same properties and could be amplified by the same system. All propagated plasmids were extracted from Top10 F`E. coli strain and subjected to enzymatic digestion by cheap restriction enzymes such as EcoRI, EcoRV, and KpnI to produce the desired size ladders [Figure 1a and b].

Bottom Line: Then, PCRs with forward and reverse 100-bp primers on the resulting plasmids amplify the ladder fragments.Moreover, 100-1500 bp fragments were produced during PCR using only a set of forward and reverse (100 bp) primers.The highest advantage of this cost-benefit approach is to produce different types of molecular weight markers by using an effective and short protocol.

View Article: PubMed Central - PubMed

Affiliation: Department of R and D, Institute Pasteur of Iran, 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.

No MeSH data available.