A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method

Authors

  • Ali Asghar Karkhane Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
  • Bagher Yakhchali Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
  • Bijan Bambai Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
  • Fatemeh Rahimi Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
  • Ferdous Rastgar Jazii Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
  • Saeed Aminzadeh Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract:

Background: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. Objectives: We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR.   Materials and Methods: Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. Results: In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. Conclusions: By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.

Upgrade to premium to download articles

Sign up to access the full text

Already have an account?login

similar resources

a nested-splicing by overlap extension pcr improves specificity of this standard method

background: splicing by overlap extension (soe) pcr is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function.objectives: we introduced a nested-soe-pcr (n –soe-pcr) in order to increase the specificity and generating mutations in a gene by soe-pcr.  materials and methods: genomic dna from bacillus thermoca...

full text

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.

BACKGROUND Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. OBJECTIVES We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. MATERIALS AND METHODS Genomic DNA from Bacillus thermoc...

full text

Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully ca...

full text

solution of security constrained unit commitment problem by a new multi-objective optimization method

چکیده-پخش بار بهینه به عنوان یکی از ابزار زیر بنایی برای تحلیل سیستم های قدرت پیچیده ،برای مدت طولانی مورد بررسی قرار گرفته است.پخش بار بهینه توابع هدف یک سیستم قدرت از جمله تابع هزینه سوخت ،آلودگی ،تلفات را بهینه می کند،و هم زمان قیود سیستم قدرت را نیز برآورده می کند.در کلی ترین حالتopf یک مساله بهینه سازی غیر خطی ،غیر محدب،مقیاس بزرگ،و ایستا می باشد که می تواند شامل متغیرهای کنترلی پیوسته و گ...

طراحی و ایجاد موتاسیون در ژن wbk A بروسلا آبورتوس S19 به روش Overlap Extension PCR

زمینه مطالعه: ایجاد موتاسیون در جایگاه اختصاصی می‌تواند یکی از روش‌های کارآمد جهت بررسی ویژگی و خواص تنظیمی ژن‌های گوناگون باشد. ﺑﺮوﺳﻠﻮز از ﻣﻬﻢ ﺗﺮﯾﻦ ﺑﯿﻤﺎریهای ﻋﻔﻮﻧﯽ ﻣﺸﺘﺮک ﺑﯿﻦ اﻧﺴﺎن و دام اﺳﺖ ﻛﻪ ﻣﻨﺠﺮ ﺑﻪ بروز ﺿﺮرهاى اﻗﺘﺼﺎدى ﻓﺮاواﻧﻰ ﻣﻰ ﺷﻮد. ﺑﻨﺎﺑﺮاﯾﻦ ﺷﻨﺎﺳﺎﯾﯽ ﻋﻮاﻣﻞ پاتوژن و اﯾﻤﻨﯽ زا در جنس ﺑﺮوﺳﻼ ﺑﻪ ﻋﻨﻮان راهگشای ﺟﻬﺖ ﮐﻨﺘﺮل اﯾﻦ ﻣﻌﻀﻞ ﺑﻬﺪاﺷﺘﯽ ﻣﻄﺮح ﻣﯽ‌ﺑﺎﺷﺪ. هدف: با توجه به اهمیت جهش هدفدار در شناسایی ساخ...

full text

A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.

A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification...

full text

My Resources

Save resource for easier access later

Save to my library Already added to my library

{@ msg_add @}


Journal title

volume 13  issue 2

pages  56- 59

publication date 2015-06-01

By following a journal you will be notified via email when a new issue of this journal is published.

Hosted on Doprax cloud platform doprax.com

copyright © 2015-2023