Ansorge W, Barker R
J Biochem Biophys Methods. 1984 Mar;9(1):33-47. doi: 10.1016/0165-022x(84)90064-2.
A system capable of resolving about 500 bases is of interest for sequencing of longer DNA molecules. Studies on further optimization of resolution on DNA sequencing gels were carried out. The effect of physico-chemical properties of gels and buffers on resolution were tested, e.g. ionic strength and pH of buffers, different buffer systems, acrylamide concentration, crosslinker concentration, type of crosslinker, temperature of polymerization, denaturing conditions, gel length and thickness. Tested were as well different running conditions like electric field, gel temperature, dimension of sample slots. Gels 0.1-0.2 mm thick and up to 1.2 m long were cast and tested routinely. Gel lengths of 60-70 cm (for sequencing up to 350-400 bases) to about 100 cm (above 400 bases) are practicable. Little is gained in resolution by increasing the gel length from 1 to 1.2 m. Resolution was improved using 0.1 mm thick gels, at a higher pH value of 8.6-8.8, and molarity increased to 0.2 M. The sequencing pattern in the region of higher bases could be better resolved on a twice-magnified picture of that region on the autoradiogram. With the long gels (70-120 cm), it is advantageous to obtain the sequence overlap by running in parallel gels of different concentrations, without re-application of samples, all loaded at the same time. Buffer chamber for running of two of three gels and thermostating plates up to 1.2 m long were designed. In this way four to six thermostated gels can be run from a power supply with two inputs. Three 1 m long gels (concentrations: 4%, 6%, 12-16%) are loaded with several samples of DNA to be sequenced and run in parallel without re-application of the samples. With good samples, the sequence overlap from the gels could be counted up to 500 base pairs, with exceptionally good samples closer to 600 bases. At present this number seems to be near the limit of the resolving power of the polyacrylamide gels.
对于较长DNA分子的测序而言，一个能够分辨约500个碱基的系统很有意义。开展了关于进一步优化DNA测序凝胶分辨率的研究。测试了凝胶和缓冲液的物理化学性质对分辨率的影响，例如缓冲液的离子强度和pH值、不同的缓冲液系统、丙烯酰胺浓度、交联剂浓度、交联剂类型、聚合温度、变性条件、凝胶长度和厚度。还测试了不同的运行条件，如电场、凝胶温度、样品槽尺寸。常规浇铸并测试了厚度为0.1 - 0.2毫米、长度达1.2米的凝胶。60 - 70厘米（用于测序多达350 - 400个碱基）至约100厘米（超过400个碱基）的凝胶长度是可行的。将凝胶长度从1米增加到1.2米，分辨率提升不大。使用0.1毫米厚的凝胶、在8.6 - 8.8的较高pH值以及摩尔浓度增加到0.2 M时，分辨率得到了提高。在放射自显影片上该区域两倍放大的图像上，可以更好地分辨较高碱基区域的测序图谱。对于长凝胶（70 - 120厘米），通过同时运行不同浓度的平行凝胶来获得序列重叠是有利的，无需重新加样。设计了用于运行三块凝胶中的两块的缓冲室以及长达1.2米的恒温板。通过这种方式，从具有两个输入的电源可以运行四到六块恒温凝胶。将三块1米长的凝胶（浓度分别为4%、6%、12 - 16%）加载多个待测序的DNA样品并平行运行，无需重新加样。对于质量好的样品，凝胶的序列重叠可达500个碱基对，对于质量特别好的样品接近600个碱基。目前这个数字似乎接近聚丙烯酰胺凝胶分辨能力的极限。
