Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

نویسندگان

  • Alexander Ulrich
  • Kasper R. Andersen
  • Thomas U. Schwartz
چکیده

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion

Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the ...

متن کامل

Application of a Seamless and Restriction Endonuclease-free Cloning Method to Produce Recombinant Full-length N-terminal His-tagged Streptolysin O in E.coli

Background and Aims: DNA cloning, sub-cloning and site directed mutagenesis are the most common strategies in nearly all projects of recombinant protein production. The classical method of restriction site cloning is unsatisfactory due to the need for supply of restriction enzymes and the inefficiency of the digestion reaction. Many new methods, including recombinatorial cloning and ligation in...

متن کامل

Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

BACKGROUND DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. RESULTS A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a s...

متن کامل

QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids

BACKGROUND Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with pe...

متن کامل

Microsoft Word - 4260_1_unknown_upload_61113_3kxf3j

Molecular cloning methods based on primer and overlap extension PCR are widely used due to their simplicity, reliability,low cost and high efficiency. In this article, an efficient mega primer (MP)-mediated cloning strategy for chimeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: i) the fir...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 7  شماره 

صفحات  -

تاریخ انتشار 2012