Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection.
BACKGROUND
Methodologies like phage display selection, in vitro 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.


RESULTS
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.


CONCLUSIONS
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.
Pepers
Barry A
BA
Center for Human and Clinical Genetics, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands. b.a.pepers@lumc.nl
Schut
Menno H
MH
Vossen
Rolf Ham
RH
van Ommen
Gert-Jan B
GJ
den Dunnen
Johan T
JT
van Roon-Mom
Willeke Mc
WM
D016000:Cluster Analysis; D016172:DNA Fingerprinting; D004797:Enzyme-Linked Immunosorbent Assay; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
D019151:Peptide Library