Construction of Tandem Repeats of DNA Fragments by a Polymerase Chain Reaction-Based Method

Abstract

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71 bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.

Unusual mtDNA Control Region Length Heteroplasmy in the COS-7 Cell Line

“…This outcome raised a question of whether multiple amplification products were an artifact of amplification. Indeed, the PCR amplification of tandem repeats can result in products with a reduced number of repeat units and under certain conditions can lead to an increase in the number of repeats [24,25]. Due to historical remoteness, DNA samples from the donor animal used to derive the CV1 cell line were not available for our studies.…”