T-RFLP技术的优缺点

T-RFLP（末端限制性片段长度多态性）该技术在应用的过程中，肯定需要在实验条件上不断进行改进，而这种改进的好坏自然而然需要实验结果的验证。

V. Grüntzig在2002年做了该工作的一部分，结果认为，在限制性酶切时，很有必要去除其中影响DNA的酶切过程，并且实验证明了具体的酶切时间。

具有说服力的结果如下：

1、T-RFLP出数据的速度快，不过只是具有半定量性；

2、DNA片断的分离有两种常用的方法：凝胶电泳，近来使用的比较广泛，且在各种实验手册中见到。毛细管电泳。

3、由于一般的实验程序无法满足毛细管实验的要求，因此，作者对该实验程式进行了改进补充。

4、实验步骤：将原文步骤贴出来，希望对大家有所帮助。

1) Extraction of community DNA from the sample of interest (soil, sediment, reactor material, water sample, etc). The DNA extraction method of user's choice should yield DNA of high quality lacking inhibitory compounds that could interfere with the subsequent PCR reaction.

2) PCR amplification of gene of interest with fluorescently labeled primers. The T-RFLP method can be applied to any gene, therefore the primer selection will depend on the user's interest. However, 16S rDNA is frequently the gene of choice for microbial community analysis (see PCR protocol). Protocols for specific genes of interest are available elsewhere in literature. The amplification can be performed using a fluorescently labeled forward or reverse primer. Both primers can also be labeled with a different dye and used simultaneously in the same reaction. It might be necessary to pool several PCR reactions to obtain enough product for further steps (200-300 ng of DNA recommended per restriction digest). The amplification efficiency of labeled primers tends to be lower than that of unlabeled primers, frequently leading to lower yields.

3) Concentration and cleaning of PCR products. Excess primers, salts and possible nonspecific PCR products are removed by gel purification. The volume of the pooled PCR reactions can be reduced to half to a fifth of the original volume using a Speedvac or ethanol precipitation in order to facilitate the loading of the sample. Gel purification is then performed by separating the PCR products by electrophoresis on agarose gel, excising the band of proper size and recovering the PCR product with a gel purification kit (Qiagen, MOBIO, Promega) following manufacturer's recommendations. If nonspecific PCR products are not detected, regular PCR clean up kits can also be used.

4) Restriction digestion of the PCR products. Once purified, the PCR products are digested with a restriction enzyme (a four base pair cutter is most appropriate as the probability of having a restriction site within the amplicon is high). Various restriction enzymes can be used in single-enzyme reactions in order to determine which one yields the highest number and most even distribution of terminal restriction fragments. For each digestion, 100-150 ng of purified PCR product (assuming a 50% loss during purification) and 10-20 U of restriction enzyme should be used. The incubation period at the enzyme's optimal temperature can vary from 4-12 h to assure complete digestion. Restriction enzymes are inactivated by heating to 65-80ºC for 20-25 min.

5)Desalting of restriction digest. In capillary electrophoresis the injection of DNA samples can be achieved by two methods. First, hydrodynamic injection requires pressure difference over the capillary. Alternatively, electrokinetic injection uses a combination of electrophoresis and electroendosmosis to inject the sample. Applied Biosystems PRISM 310 and 3100 Genetic Analyzers (PE Biosystems) use the latter. The presence of ions can interfere with the uptake of DNA using electrokinetic injection because of preferential injection of higher charge-to-mass molecules (e.g. Cl¯ ions). Therefore it is essential to desalt the inactivated restriction digest with Microcon columns (Amicon), Qiaquick Nucleotide Removal Kit (Qiagen) or conventional ethanol precipitation. In our case, the restriction products were diluted with water up to 500 μl, before concentration and desalinization on Microcon columns (see figure below).

6) Capillary electrophoresis (CE); loading. A fifth to a third of the desalted restriction digest is generally loaded onto the capillary electrophoresis system using the default settings optimized for sequencing. This can lead to insufficient fluorescent signal, therefore yielding a low number and height of peaks. The injection time and injection voltage can be varied to regulate DNA uptake. The default sample injection time is generally 10 sec, however longer injection times increase the uptake of DNA from dilute sample solutions, therefore yielding a higher fluorescent signal with more peaks detected. Furthermore, injection voltage is directly proportional to the amount of DNA injected and can be modified from the default of 3 kV. Further concentration of the desalted product may also be necessary to stay within the appropriate CE loading volume.