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Optimizing the Direct Amplification of Missing cDNA 5' Ends,,, Using the Eppendorf Mastercycler gradient

Optimizing the Direct Amplification of Missing cDNA 5' Ends
Using the Eppendorf Mastercycler gradient

Ulrich Genschel
Institute of General Botany, University of Hamburg Christian Rohrer, Eppendorf AG, Hamburg

Introduction

In many research projects, cDNA isolation forms the basis for detailed analyses at the molecular level. However, when cDNA libraries are screened using conventional plaque hybridization techniques, cDNA clones with an incomplete 5' end are frequently obtained. Since a complete cDNA, or at least a complete open reading frame, is required for many applications (such as expression of functional proteins or sequence-based prediction of sub-cellular location), the missing 5' sequence has to be obtained at a later point in time. However, isolation of full-length cDNAs by plaque screening is extremely labor-intensive when the sought-after cDNA is present at low abundance in a given library.

Direct amplification

One alternative is direct PCR amplification of the missing sequence using the cDNA library as template [1,2]. In this case, a vector primer (VP) and a gene-specific primer (GP) are created as shown in the scheme in Fig. 1. As all cDNA clones in the library act as a template for the vector primer, nonspecific products are formed very easily in this PCR reaction. It is therefore important to use highly-specific primers and to ensure that reaction conditions are as stringent as possible, with one crucial factor being the setting of the annealing temperature. An example of this strategy is given below.


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