Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

• Main Authors: van Ommen Gert-Jan B, Vossen Rolf HAM, Schut Menno H, Pepers Barry A, den Dunnen Johan T, van Roon-Mom Willeke MC
• Format: Article
• Language: English
• Published: BMC 2009-05-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/9/50

<p>Abstract</p> <p>Background</p> <p>Methodologies like phage display selection, <it>in vitro </it>mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.</p> <p>Results</p> <p>HRMA results nicely matched those obtained with ELISA and compared favourably to DNA fingerprinting of restriction digested clone insert-PCR. DNA sequence analysis confirmed that HRMA-clustered clones contained identical inserts.</p> <p>Conclusion</p> <p>Using HRMA, analysis of up to 384 samples can be done simultaneously and will take approximately 30 minutes. Clustering of clones can be largely automated using the system's software within 2 hours. Applied to the analysis of clones obtained after phage display antibody selection, HRMA facilitated a quick overview of the overall success as well as the identification of identical clones. Our approach can be used to characterize any clone set prior to sequencing, thereby reducing sequencing costs significantly.</p>