Amplicons cloning

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [>600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

Figure 2.2 Schematic diagram of in vitro transcription reaction. The telomerase RNA gene is cloned behind a T7 RNA promoter. PCR is used to generate linear DNA and the PCR amplicons are in vitro transcribed with T7 RNA polymerase. The RNA is purified away from free nucleotide on a desalting column.

The relatively low cost of cDNA array production and the access to thousands of EST clones in the freezers in many laboratories and the commercial distribution of EST clone collections propelled the early development and popularity of the cDNA arrays in the late 1990s (15). The arrays are generated through PCR-amphfication of cloned 200 000 bp insert sequences using vector-specific primers. The double-stranded amplicons are... 

Frenkel N, Spaete RR, Vlazny DA, Deiss LP, Locker H (1982) The herpes simplex virus amplicon—a novel animal-virus cloning vector. hr Eucaryodc Viral Vectors (Gluzman Y, ed), pp 205—209. New York Cold Spring Harbor Laboratory. 

Spaete RR, Frenkel N (1982) The herpes simplex virus amplicon A new eucaryodc defecdve-vuus cloning-amplifying vector. Cell 30 305-310. 

Ray, M., Guan, X., Slovak, M Trent, J., and Meltzer, P. (1994) Rapid detection, cloning and molecular cytogenetic characterization of sequences from an MRP-encoding amplicon by chromosome microdissection. Br. J. Cancer 70, 85-90. 

PCR products from 92 amplicon-positive isolates were cloned and sequence data were obtained from at least one clone per isolate approximately 2/3 of the isolates (66 total) contained PKS sequences based on inferred amino acid homology. The remaining sequences from 26 isolates were not representative of KS domains and did not correspond with any single class of genes by blastx analysis. 

Cloning and sequencing of amplicons. The amplified products were cloned into the pGEM-T vector (Promega) and used to transform competent E. coli DH5a. Transformed E. coli DH5 was then plated onto Luria-Bertani agar with 50 pg/mL... 

Figure 26 Compartmentalized self-replication. The polymerase gene library is cloned into an expression vector and subsequently transformed into an E. coli cell. After the polymerase library is expressed, the cells are isolated and encapsulated with PCR reagents using a water and oil emulsion. The emulsion is then thermocycled, destroying the E. co//cell wall and allowing amplification to occur. Post-CSR, the amplicons enriched in genes encoding active mutants can be collected and purified for the next round of selection.

Fig. 3a). DNA extracted from the clone that corresponds to the background control transformation, which contains only YAC, is used as a positive control for the native-specific amplicons and a negative control for the synthetic-specific amplicons. 

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