Agencourt offers a full array of library construction services including:

  • Standard cDNA Libraries
  • Large insert cDNA Libraries
  • Normalized cDNA Libraries
  • Subtracted cDNA Libraries

We use unique processes at every stage of cDNA library construction including:
  • Isolation of highly pure cytoplasmic mRNA
  • Proprietary cDNA synthesis methodology
  • Proprietary clamped dT primer technology
  • Novel cloning methods to retain larger clones

Maximum Flexibility
Our cDNA libraries are constructed from tissues, cells or purified total RNA and directionally cloned into Agencourt's custom vectors or customer-supplied vectors. This flexibility allows us to produce libraries that meet your specific application needs. Methods utilized to construct these may include but are not limited to the publications listed below.

Extensive Experience
Agencourt takes part in cDNA library construction, 5' EST sequencing and cDNA finishing for the Mammalian Gene Collection (MGC) project. We have sequenced over 2.5 million EST sequences and greater than 20,000 full length cDNA sequences to the MGC project.


Standard and Large Insert Libraries

Innovative Technology
Agencourt's innovative technology combines optimized cDNA synthesis methods and novel quality control processes to maximize library quality. Our technology produces cDNA libraries with (Table 1):
  • Increased number of full length clones
  • Larger number of primary clones
  • Increased number of recombinant clones

  


  Standard Large Insert
Starting Material Total RNA (≥1mg)
Cells (≥1.0 x1 08)
Tissue (≥1g)		 Total RNA (≥2mg)
Cells (≥2.0 x 108)
Tissue (≥2g)
Primary Clones 2 x 107 1 x 107
Avg. Insert Size >1kb >3kb
% Recombinants >95% >95%
Table 1: Properties of typical Standard and Large Insert cDNA Libraries.

Successful Cloning Approaches
Agencourt employs optimized normalization techniques to reduce the frequency of highly abundant clones and increase the frequency of rare clones. Our normalized libraries contain a high number of recombinant clones that retain the average insert size of the primary library. A typical normalized library has a greater than 100-fold reduction in highly abundant clones, such as gamma-actin (Table 2). Subtracted libraries are available for customers interested in identifying unique gene expression patterns. This technology enables rapid characterization of genes differentially expressed between two populations, such as drug-responsive versus unresponsive cells.

  


  Primary
Library		 Normalized
Library
No. Primary Clones 2.5 x 107 1.2 x 107
Avg. Insert Size 2.38kb 2.35kb
Insert Range 1.2 - 5kb 0.5 - 6kb
% Recombinants 100% 100%
% Actin Level 0.67% 0.005%
Table 2: Primary and normalized cDNA libraries were constructed from Human Glioblastoma cells. Actin levels were determined using colony hybridization.

Decreased Sequencing Time and Cost
Agencourt's cDNA synthesis and cloning processes lead to a high frequency of full length clones. This feature decreases sequencing costs by reducing the number of reads required to obtain complete gene sequences. Additionally, Agencourt has a comprehensive genomic pipeline (Figure 1) that allows our clients to seamlessly move from library construction into high throughput DNA sequencing. Our cDNA sequencing and finishing service provides for optimum cDNA project turnaround, while ensuring the highest level of DNA sequence quality.

  



Learn more about our DNS Sequencing Pipeline
Figure 1: Agencourt's comprehensive pipeline is integrated using an Oracle-based LIMS. Our Galaxy LIMS™ continually tracks samples and assesses pipeline performance to ensure production of the highest quality libraries and sequencing results.

Duguid, J.R.., Rohwer, R.G. and Seed, B. 1988. PNAS USA 85: 5738-5742.

Rubenstein, J.L., Brice, E.J., Ciaranello, R.D., Denney, D., Porteus, M.H. and Usdin, T.B. 1990. Nucleic Acids Res. 18: 4833-4842.

Wieland, I., Bolger, G., Asouline, G. and Wigler, M. 1990. PNAS USA 87:2720-2724.

Swaroop, A., Xu, J., Agarwal, N. and Weissman, S.M. 1991. Nucleic Acids Res. 19:1954.

Swaroop, A., 1993. Methods in Neurosciences 15: 285-300.

Gruber, C.E., Li, W.B., Lin, J.-J. and D'Alessio, J.M. 1993. Subtractive cDNA Hybridization using the Multifunctional Plasmid Vector pSPORT 2. Focus 15, 59-65.

Li, W.B., Gruber, C.E., Lin, J.-J., Lim, R., D'Alessio, J.M. and Jessee, J. 1994. The Isolation of Differentially Expressed Genes in Fibroblast Growth Factor Stimulated BC3H1 Cells by Subtractive Hybridization. BioTechniques 16, 722-729.

Gruber, C.E. and Li, Wu-Bo. 1995. An Improved Subtractive Hybridization Method using Phagemid Vectors. "Molecular Biology: Current Innovations and Future Trends", p.93-106 (Eds. A. Griffin & H. Griffin) Horizon Scientific Press, U.K.

Li, Wu-Bo and Gruber, C.E. 1996. An RNA Subtraction Method for Library Construction. "A Laboratory Guide to RNA: Isolation, Analysis and Synthesis", p.323-338 (Ed. P. Krieg) Wiley-Liss, Inc., N.Y., N.Y.