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2014 ; 27
(10
): 301-7
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From deep sequencing to actual clones
#MMPMID25183780
D'Angelo S
; Kumar S
; Naranjo L
; Ferrara F
; Kiss C
; Bradbury AR
Protein Eng Des Sel
2014[Oct]; 27
(10
): 301-7
PMID25183780
show ga
The application of deep sequencing to in vitro display technologies has been
invaluable for the straightforward analysis of enriched clones. After sequencing
in vitro selected populations, clones are binned into identical or similar groups
and ordered by abundance, allowing identification of those that are most
enriched. However, the greatest strength of deep sequencing is also its greatest
weakness: clones are easily identified by their DNA sequences, but are not
physically available for testing without a laborious multistep process involving
several rounds of polymerization chain reaction (PCR), assembly and cloning.
Here, using the isolation of antibody genes from a phage and yeast display
selection as an example, we show the power of a rapid and simple inverse
PCR-based method to easily isolate clones identified by deep sequencing. Once
primers have been received, clone isolation can be carried out in a single day,
rather than two days. Furthermore the reduced number of PCRs required will reduce
PCR mutations correspondingly. We have observed a 100% success rate in amplifying
clones with an abundance as low as 0.5% in a polyclonal population. This approach
allows us to obtain full-length clones even when an incomplete sequence is
available, and greatly simplifies the subcloning process. Moreover, rarer, but
functional clones missed by traditional screening can be easily isolated using
this method, and the approach can be extended to any selected library (scFv,
cDNA, libraries based on scaffold proteins) where a unique sequence signature for
the desired clones of interest is available.
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