Publications

This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them.

The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells.

BACKGROUND

Effective communication between referring and specialty providers is key to optimizing patient safety. Communication was assessed in an electronic referral system by review of referrals to a public urban health care system's gastroenterology clinic that were not scheduled for appointments.

METHODS

All electronic referrals to a publicly funded, urban health care system's adult gastroenterology clinic from November 1, 2009, to November 30, 2010, were reviewed that did not result in scheduling of appointments. An assessment was made of whether in-person visits were unnecessary by preconsultation exchange or whether the referrals remained unscheduled for other reasons. For the latter group, reasons why the referrals remained unscheduled were examined, and medical records were reviewed for actual patient harm when sufficient information was present in the chart or for potential harm when no further information about the referral complaint was available.

RESULTS

Eighty-six (32%) of 266 not-scheduled referrals were resolved via preconsultation exchange. For another 96 (36%), patients were not ultimately considered to require appointments or were scheduled via other routes. Nine patients received unplanned care while awaiting scheduling decisions, 5 of whom had harm that was related to referral complaints, although scheduling of appointments may not have avoided this harm. Of 75 patients for whom further information was not available about the referral complaints, most were not seen back in primary care, and 55 (73%) had potential for major harm.

CONCLUSION

Few adverse outcomes in electronic referrals not scheduled for in-person gastroenterology visits were found, and none were clearly due to communication lapses in the referral process. Contributors to the potential for harm in referrals that were unintentionally left unscheduled included discontinuity of care and lack of patient or provider follow-up.