Rapid Directional Walk Within DNA Clones by Step-Out PCR
Genomic segments of many model experimental organisms are now available as segments within cosmid, P1, or YAC vectors (1 –3 ). Even cDNA portions of these segments are becoming available. Preliminary analyses with any of these clones would include sequencing and mapping (either on the genome or within a set of clones or subclones). Generally, such analysis would begin with sequences at the termini of inserts and proceed directionally into the internal sequences. PCR, with one specific primer and one nonspecific primer, is an ideal procedure to generate sequential and ordered fragments for use as sequencing templates or probes in nucleic acid hybridizations (4 ). These fragments would extend from a specific position within the known sequences of either the vector arm or the insert (via the specific primer) into different positions within the adjacent unknown sequence (via the nonspecific primer). This “Step-Out PCR” procedure generates fragments in a matter of h and eliminates the need for time-consuming procedures, such as restriction enzyme digestion/analysis, ligations, and transformation into hosts (5 –8 ).
- Chemokine Overexpression in the Skin by Biolistic DNA Delivery
- Molecular Modeling and Simulation of G-Quadruplexes and Quadruplex-Ligand Complexes
- Atomic Force Microscopy and High-Content Analysis: Two Innovative Technologies for Dissecting the Relationship Between Epithelia
- Derivation of Induced Pluripotent Stem Cells by Retroviral Gene Transduction in Mammalian Species
- Systemic Delivery of Antisense Oligomer in Animal Models and Its Implications for Treating DMD
- Preparation and Analysis of Drosha
- Construction of Plasmids Containing Site-Specific DNA Interstrand Cross-Links for Biochemical and Cell Biological Studies
- Construction of Simple and Efficient siRNA Validation Systems for Screening and Identification of Effective RNAi-Targeted Sequen
- Direct Use of Phage Particles for DNA Transfection
- Detection of DNA Damage by Comet Fluorescence In Situ Hybridization