Hannon K, Johnstone E, Craft L S, Little S P, Smith C K, Heiman M L, Santerre R F
Department of Biotechnology Research, Lilly Research Laboratories, Indianapolis, Indiana 46285.
Anal Biochem. 1993 Aug 1;212(2):421-7. doi: 10.1006/abio.1993.1350.
We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.
我们报道了聚合酶链反应（PCR）衍生的单链DNA（ssDNA）探针的新型合成方法及其在原位杂交中的后续应用。将妊娠11.5天小鼠胚胎的连续横切片与对应于肌细胞生成素cDNA中相同181碱基对序列的33P-ssDNA、33P-RNA或35S-RNA探针杂交。使用33P-ssDNA获得的信号比使用33P-RNA探针获得的信号更强，而两种33P探针获得的信噪比远优于35S探针。与鸡垂体切片杂交时，地高辛标记的鸡生长激素（GH）ssDNA产生的信号比地高辛标记的鸡GH RNA产生的信号略强。发现32P-ssDNA探针适用于Northern印迹杂交。使用ssDNA探针进行原位杂交的优点包括：（1）ssDNA技术快速且简单。无需将DNA模板克隆到特殊的RNA载体中或订购特殊的含T7的PCR引物。使用实验室现有的任何引物，可在不到1天的时间内合成ssDNA探针（原位杂交的最佳探针长度为50至200碱基对）。（2）在三个独立的原位实验中，ssDNA探针产生的信号比RNA探针更强。（3）ssDNA探针可能比RNA探针更稳定。（4）由于省去了RNA酶冲洗步骤，与ssDNA探针杂交时杂交后冲洗步骤缩短。通过该方案产生的ssDNA探针可用多种不同的同位素（放射性和非放射性）进行标记，是用于原位杂交的优良探针。
