The primary interest of my lab is protein engineering. We have focused this interest mostly on the engineering of high fidelity restriction enzymes. Many restriction enzymes tolerate a degree of ‘wobble’ in their recognition sequences and as a result they cut DNA at additional, off-target, sites. This unwanted cleavage activity, first observed with EcoRI more than 30 years ago, is termed ‘star-activity’. The molecular basis for star-activity varies from enzyme to enzyme, and becomes more apparent during digestions at high enzyme or glycerol concentrations, or for extended incubation times. Star activity is minor for many restriction enzymes, but for some it can be very problematic, radically altering digestion patterns. Using a systematic engineering and screening method, my lab has succeeded in isolating improved forms of many problem enzymes, forms that display much-reduced or no detectable star-activity. We call these ‘high-fidelity’ (HF) restriction enzymes. Currently, 31 such HF enzymes are available from NEB: BamHI-HF, NotI-HF, KpnI-HF, SacI-HF, SalI-HF, PstI-HF, PvuI-HF, ScaI-HF, SspI-HF, HindIII-HF, NcoI-HF, NheI-HF, SpeI-HF, EcoRV-HF, BstEII-HF, NcoI-HF, BsrGI-HF, EcoRI-HF, EagI-HF, SphI-HF, StyI-HF, StyI-HF, SbfI-HF, BmtI-HF, PvuII-HF, BsaI-HF, DraIII-HF, AgeI-HF, MfeI-HF, MluI-HF, and NruI-HF. More will be available soon.
The second interest of our lab is on reverse transcriptase and its application in low-input RNA sequencing. Low-input RNA sequencing has been widely used in single cell analysis, which is revolutionizing our understanding of the enormous diversity of the transcriptome in both normal and pathological states. To optimize low-input RNA sequencing technology, we carry out different approaches including protein engineering with the aim to increase reverse transcription efficiency, attenuate amplification distortion, reduce false positive errors and increase coverage uniformity.