We first optimized the LASL method (Figure 1) by reducing template contamination during shearing, and taking steps to minimize cloning biases, particularly for AT-rich templates.
This is logical, as longer extension times reduce template switching, providing less opportunity for crossovers to occur.
By eliminating RT and using exon-specific barcodes to ensure unambiguous isoform assignment, SeqZip should greatly reduce template switching.
A single 3′ adenosine was added to the repaired ends using Klenow exo- and dATP to reduce template concatemerization and adapter dimer formation, and to increase the efficiency of adapter ligation.
Theoretically, increasing primers' binding strength may also increase the sensitivity of conventional PCR, reducing minimum template requirements.
Gel extraction can reduce the template background.
Furthermore, the product mixture of MegaWHOP must be digested by a dam-methylated DNA-specific restriction enzyme, such as DpnI, to reduce the template plasmid.
For microarray analysis, RNA aliquots of 4 controls and 4 affected samples were pooled to reduce the template needed and to exclude differences in expression due to individual variation and not related to BSP [ 5].
Matsumoto indicated that one toxic function of Al in rapidly growing pea roots was the binding of Al to DNA regions unmasked with chromosomal proteins in nuclei causing the condensation and stabilization of chromatin structure and thereby reducing the template activity.
