Navigation Links
Cloning Based on Efficient Three-Fragment Assembly,,,DNA Ligation


The efficiency of the Rapid DNA Ligation Kit from Roche Molecular Biochemicals was compared with standard and optimized conventional ligation procedures. The reactions generated circular recombinant bacteriophage DNA in a three-fragment assembly DNA ligation. Sequential blunt-end and cohesive-end ligation, including a temperature shift from 26C to 14C, led to a more than 10-fold increase in the number of transformants obtained. Applying a modified protocol, the kit was found to provide a reliable, fast, and remarkably efficient procedure for this complex application.


To construct a phage displaying integrin-binding ligands, we needed a reliable and efficient ligation procedure, by which three DNA fragments could be assembled simultaneously, yielding circular DNA. Such a ligation is known to require high concentrations of the reacting DNA molecules [1], especially if blunt-ended DNA is involved [2] and is therefore usually circumvented by subcloning. Because of the absence of appropriate restriction sites in the DNA fragments, subcloning into another suitable vector and subsequent ligation of the two fragments in this construct was impossible in our case. A standard ligation protocol led only to poor transformation results, whereas parallel ligations using either an optimized conventional procedure or the Rapid DNA Ligation Kit from Roche Molecular Biochemicals resulted in efficient and correct formation of the desired cloning vector.

Materials And Methods

The bacteriophage fd-tet which confers tetracycline resistance upon infected cells was donate d by Dr. John McCafferty (Cambridge Antibody Technology).
Two DNA fragments (here designated as fragments A and B) had to be joined at a common unique Alu I site and ligated with a fd-tet DNA backbone at Apa LI and Bam HI restriction sites (Figure 1). Both insert fragments were obtained by cleavage of cloned DNA with the endonucleases Apa LI, Bam HI, and Alu I. The Apa LI/Alu I fragment of insert A and the Alu I/Bam HI fragment of insert B were purified after separation on an agarose gel. One microgram of DNA in a total volume of 10 l was used in the conventional ligation reactions, with a molar ratio of phage DNA to insert A and insert B of 1:5:5. Standard ligations of the two insert fragments together with appropriately cleaved fd-tet DNA were performed for 24 h at 14 C. Our optimized procedure was comprised of two temperature steps: first, the blunt-end ligation of the insert fragments A and B for 18 h at 26C; second, the cohesiveended vector DNA was added, and the ligation continued for an additional 6 h at 14C [3]. The protocol for the Rapid DNA Ligation Kit was modified in accordance with our optimized conventional procedure. The blunt-end ligation mixture (290 ng DNA in a total volume of 20 l) was ligated for 30 min at 26C. Next, 710 ng vector DNA was added in a volume of 1 l with further incubation for 10 min at room temperature.

The ligation mixtures were then cooled to 0C without heat-inactivation of the ligase, and one third of each reaction was used to transform DH5a competent cells, which were plated on 140 mm Petri dishes containing 30 mg tetracycline/ml LB medium. After a 20 h incubation period, colonies were counted, and doublestranded phage DNA of a representative number purified with the High PureTM Plasmid Isolation Kit. The DNA was screened by restriction analysis with the endonuclease Sca I, followed by sequencing of all restriction-positive clones using the primer 5'-TGAATTTTCTGTATGAGG-3', the T7 Sequenase 2.0 kit, and 33PdATP from Amersham, according to the manufacturers instructions.

Results and Discussion

Although a T4 DNA ligase-overexpressing E. coli strain that allows cloning of linear DNA molecules was reported recently [4], efficient transformation of E. coli normally requires circular DNA. However, the formation of a circular structure in a three-fragment assembly ligation is a relatively rare event. At high DNA concentrations, which are crucial for a three-component assembly ligation [1], predominantly intermolecular ligation events leading to linear products occur, while DNA circularization is favored at low concentrations of the DNA fragments [5]. Our case was further complicated by the need to ligate two blunt-ended DNA fragments. Blunt-end ligation is known to be one to two orders of magnitude less efficient than cohesive-end ligation [6]. We chose to optimize the conventional procedure by sequential ligation of blunt- and cohesive DNA ends using different temperatures for the two processes. Therefore, we started the reaction at 26C, which was reported to be the optimal temperature for the joining of bluntended termini by T4 DNA ligase [7]. We followed this with cohesive-end ligation and circularization of our DNA at 14C, the temperature generally regarded as the best compromise to retain sufficient activity of the enzyme while still allowing relatively stable hydrogenbonded association of cohesive termini [8]. This strategy led to a more than 10-fold increase in the number of transformants obtained compared with a control ligation performed at single temperature (Table 1).

Using a protoco l for the Rapid DNA Ligation Kit that was still suboptimal with respect to DNA concentration and recommended dephosphorylation of the vector DNA, the yield of transformants was 5-fold higher than with the optimized conventional ligation procedure. This remarkably high efficiency of the kit is most likely due to an optimized composition of the ligation buffer.

Although, in theory, 100% correct clones can be obtained by a strategy involving fragments that are designed to join in the desired order and orientation, a notable number of false-positive clones arose in all our ligation experiments. Most of them were due to contamination resulting from incompletely cleaved and self-ligated phage DNA backbone. A relatively small proportion of deletion mutants and clones containing a doubled phage backbone was also found.

The excellent performance of the Rapid DNA Ligation Kit was reproducible with different insert pairs. Thus, the kit provides a reliable and remarkably efficient way for the three-fragment assembly DNA ligation to generate circular DNA under minimal time requirements.



Page: All 1 2 3 4

Related biology technology :

1. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
2. Enhanced PCR Cloning System
3. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
4. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
5. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
6. QIAGEN PCR CloningPlus Kit
7. Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments
8. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
9. Development of a Multiplex Bead-Based Assay for Antibody Screening of a Nonhuman Primate Colony on the Bio-Plex System
10. A Mix-and-Read Cell-Based Assay for Hybridoma Screening Using the FMAT 8100 HTS System
11. Mix-and-Read, Bead- and Cell-Based Immunoassays Using the FMAT 8100 HTS System
Post Your Comments:

(Date:11/24/2015)... ... November 24, 2015 , ... ... Technologies, Inc., on being named to Deloitte's 2015 Technology Fast 500 list of ... OrthoAccel manufactures AcceleDent®, a FDA-cleared, Class II medical device that speeds up orthodontic ...
(Date:11/24/2015)... ANGELES , Nov. 24, 2015 ... biotechnology company focused on the discovery, development and commercialization ... , Ph.D., Chief Executive Officer, is scheduled to present ... 1, 2015 at 10:50 a.m. EST, at The Lotte ... City . . ...
(Date:11/24/2015)... 24, 2015  PDL BioPharma, Inc. (PDL) (NASDAQ: PDLI ... company,s president and chief executive officer, will present at the ... in New York City . The presentation ... 1, 2015 at 9:30 a.m. EST. ... website at least 15 minutes prior to the presentation to ...
(Date:11/24/2015)... ... November 24, 2015 , ... Whitehouse Laboratories is pleased to announce that it ... will be strictly dedicated to basic USP 61, USP 62 and USP 51 testing ... have complete chemistry and micro testing performed by one supplier. Management has ...
Breaking Biology Technology:
(Date:11/18/2015)... PHILADELPHIA , Nov. 18, 2015  As new ... in children, doctors and other healthcare providers face challenges ... counsel families and patients. In addition, as more children ... into a patient,s adulthood and old age. ... The Children,s Hospital of Philadelphia (CHOP) . ...
(Date:11/17/2015)... -- Paris from 17 th ... Paris from 17 th until 19 th ... has invented the first combined scanner in the world which ... surface. Until now two different scanners were required: one for ... on the same surface. This innovation is an ideal ...
(Date:11/17/2015)... , Nov. 17, 2015  Vigilant Solutions announces today ... its Board of Directors. --> ... recently retiring from the partnership at TPG Capital, one ... with over $140 Billion in revenue.  He founded and ... all the TPG companies, from 1997 to 2013.  In ...
Breaking Biology News(10 mins):