????????????????????????(DNA ? RNA)????????????????????????,??????????????RNA ??(???? 16S rRNA ??)???????????????????,?????????????????,????????????????? 16S rRNA ????????????????????????(V ?)???,???????????????????????,? Ribosomal Database

1
????????????
2
??
????????
?????????
???????????
???????-??454???????
???????????
3
????????
????????????????????????,?????????????????
???,?????????????????????,????????????????????????
????????1,2,??????????????????????,?????????????
??????????????
4
?????????
?????????????????????3,4,5,6?
??????????????,????????????????????????????
?,????????????????,???????????????,???????
??????????????,????????,?????????????,???????,???
?????????????????????????????,????????????????????
???? ????????,??????????????
5
???????????(????????)
??????????,??????????????????????,??????
??????????????????,???????????????7??????????,??
???????????????????,??????????????????????????????
??? ??,????????????????????,??????????????
????????????,??????????????????????????????,?????
??????????????????????8???,????????????????,????
??????????????????,??????????????

6
???????????(?????????)
????????????????????????(DNA ?
RNA)????????????????????????,??????????????RNA
??(???? 16S rRNA ??)???????????????????,??????????
???????,????????????????? 16S rRNA
????????????????????????(V ?)???,?????????????????
??????,? Ribosomal Database Project(RDP)?SILVA
?Greengenes ??,???????????????? 16S
rRNA????????????,?????????9?
??????????????????? DNA ????????????DNA
??????????????,???????????? PCR(Real time
quantitative PCR)?????????(Fluorescence in situ
hybridization, FISH)??????????
7
???????????(?????????)
DNA ???????????????????????,????????????
? DNA ???????????????,??????????????????????,?????
????????????????DNA ??????????????????,???????????
????????????????????????? DNA ????????????????(Den
atured gradient gel electrophoresis,DGGE)10,11,12
??????????(Terminalrestriction fragment length
polymorphism, T-RFLP)?? ?????????,DNA
????????????????????????,?????????????????????????
?????????(Sanger)??????? 16S rRNA
????????????????????????????
8
???????-??454???????
????????? ?PCR??????????,??DNA????ATP??????
?????????????????????5'?????????,?????????????????
??????????????????????,?????dNTP,??dNTP???????,
DNA ????????????????????????????( PPi)
?ATP?????APS?????????????ATP,ATP??????????????????
( oxyluciferin)???,????????ATP???????????ATP?????d
NTP????????????,?????,???????,???????dNTP,????13
?
9
???????-??454???????
454?????? (1)???????? GS????????DNA?PCR??????????
(BACs) ?cDNA????RNA ?????????DNA
?BACs??????????300-800 bp ???,????????RNA ?PCR
??, ??????? (2)DNA ???????????????????????DNA???5
'???????,3'?????,?????44 bp ????(
adaptor)A?B??????????5'??3'?????A?B??????A
B?????DNA??????????
10
???????-??454???????
(3)DNA ??????????????????,?????DNA??????,????????
??AB ????????,????????????????????,???DNA?????????
???????,?1?DNA????1???? (4)??PCR???????????PCR???
?????????-??????????,????DNA ????emPCR ??PCR (
emPCR)???
11
???????-??454???????
(5)?????????, ??????DNA???????,????PCR????,??????
DNA????????,?????????????????14?
12
????
(1)??????? ????????????????8-11??????????
??????????????0d(???)?7d?14d?21d(??)?24d?28d??,???
?????? (2)?????DNA?? ?DNA????E.Z.N.A.TM
Stool DNA Kit????
? M ?? DNA marker1 ????E. Z. N. A stool
DNA??????????2????E. Z. N. A soil DNA???????????
13
????
(3)PCR??16S r RNA???? ?????????????,??16S
rRNA???V3-V6?????PCR????????????????RDP16S
???????,???????????gt98???16S????,?????????????PCR
????(????IDtag)?4????????(????)? PCR??????DNA
2µL
10Buffer(Mg2)5µL
dNTP 2µL
5????(10µM)1µL
3????(10µM)4???????1µL
Taq? 0.5µL
ddH2O 35.5µL PCR????94????4min,94??
?30s,55???30s,72???50s,?25???,??72???10min?
PCR????????????????????? (4)454?????
14
??????
(1)????? ???????????????????????????,?????
???,????????????? ??? barcode ?????,????????????
??????????? 150 bp ???? 80????????????
20 ?????????? 20 ?????? ????????,?????????
barcode ???????????? ?????????,??????????????
barcode ??,????????????? (2)??OTU????????
????????,????????,???? 97?? OTU ??,????? 97????
OTU?? OTU???????? a ?????,????OTU ??( Total
Abundance )?????( Species Richness
)???????(Shannon-Weiner),????????(Simpsons
diversity)??????(Pielous evenness),????????Chao1?
ACE???????????500???,??????(Rarefaction
curve)???????????????
15
??????
(3)???????????? ??RDP Classifier
??????RDP16S ???????????????0.8
?????,???????????????????????????????????????,????
????????????,?????????????,???????????????? (4)???
??? UniFrac ????????????????????,?????????
?????????????? UniFrac??????????????????,?????????
,?????????????
16
????

• Zoetendal E, Koike S, Mackie R, et al. Molecular
  ecological analysis of the gastrointestinal
  microbiota A review. J of Nutrition, 2004,
  134465-472.
• ???,???,????.??????????????????J.??????,1985,16
  7-72.
• Willard M D, Simpson R B, Delles E K, et al.
  Effects of dietary supplementation of
  Fructooligosaccharides on small intestinal
  bacterial overgrowth in dogs. Am. J Vet Res,
  1994, 55654-659.
• Suau A, Bonnet R, Sutren M, et al. Direct
  analysis of genes encoding 16SrRNA from complex
  communities reveals many novel molecular species
  within the humangut. Appl Environ Microbiol,
  1999, 654799-4807.
• Ward J. Hierarchical grouping to optimize an
  objective function. J Am Stat Assoc, 1963,
  58236-244.
• Watanabe K, Kodama Y, Harayama S. Design and
  evaluation of PCR primers to amplify bacterial
  16Sribosomal DNA fragments used for community
  fingerprinting. J Microbiol Methods, 2001,
  44253-262.
• ???,???,????.???????????????????????J.????????,2
  009,24(6)497-503.
• ???.???????M.???????,2004114-130.
• ??,???,???.??????????????????????????J.?????????
  ,2005,11(3)381-387.
• Muyzer G, De Waal E, Uitterlinden A. Profiling of
  complex microbial populations by denaturing
  gradient gel electrophoresis of polymerase chain
  reaction-amplified genes coding for 16S rRNAJ.
  Applied and Enviromental Microbiology,
  1993,59(3)659.
• ???,???,???.DGGE/TGGE?????????????????J.?????,20
  04,44(6)845-848.
• ???.????????(DGGE)???????????J.????,2003,23(8)1
  561-1569.
• ???. Genome Sequencer FLX??????????????.??????,200
  8,35(1)149-151.
• Rothberg J M, Leamon J H. The development and
  impact of 454 sequencing. Nature Biotechnology,
  2008, 261117-1124.

17
Thank You !