3．討論
隨著近年基因芯片技術的發展，研究者逐漸認識到基于核酸雜交原理的傳統基因芯片缺陷與應用的局限性。隨著PCR技術的進展，特別是熒光定量PCR技術的出現PCR技術已成為生物醫學領域中應用最廣泛的技術。如果一種基因芯片能直接進行PCR反應,而且能夠同時擴增大批可能發生變異的基因顯然會有廣泛的用途。我們與微電子所合作，利用其微加工能力，首次建立含有數百上千微反應池的PCR基因芯片。近年來熒光PCR方法有所發展，我們設想在如果芯片上設置多個微反應池進行熒光PCR反應，通過檢測芯片上熒光的變化來確定反應結果，可以免去電泳等繁瑣的檢測過程，并且可在不同反應池中同時進行多個反應。PCR反應在芯片內極小容積的微反應池中進行，每個微反應池均可以將其內部的基因擴增數百萬倍，解決了常規PCR一次僅僅可以分析一種或者少數幾種基因的不足。微反應池內的反應介質的量僅為普通PCR一次反應的量，也會大大節約試劑開支。
PCR基因芯片的研制面臨重要問題是在僅數十納升的微反應池中能否進行微量的熒光PCR反應？熒光的變化能否準確檢測？如何選擇用于不同用途的PCR反應類型？
我們利用臨床病例和正常人實際例證，探討利用Taqman探針和SYBR Green熒光染料在PCR基因芯片上做熒光PCR反應的可行性。
TaqMan熒光探針是特異合成的寡核苷酸序列［9］，其5’、3’分別標有熒光報告基團（如FAM）、熒光淬滅基團（TAMRA）。在PCR擴增前，TAMRA通過F?ster能量遷移（FRET）作用，抑制FAM的熒光發射。如PCR產物中有靶序列存在，則在PCR擴增的延伸階段，Taq酶通過其5’→3’外切酶活性將結合的TaqMan探針切斷，FAET抑制作用被解除，后者的熒光信號得以釋放出來，模板DNA每復制一次，就有一個熒光信號被釋放［10-11］。我們所設計的檢測遺傳性鐵幼粒細胞貧血家系G514A點突變的探針，結果證明這類反應能在PCR基因芯片上順利反應。在結果圖中可以看出陰性與陽性結果之間差別仍很明顯。但是，TaqMan熒光探針的應用仍然存在問題，大量合成TaqMan熒光探針會十分昂貴，平衡大量PCR反應的條件也是很困難的事。我們又試用熒光染料SYBR
Green。SYBR
Green可以與雙鏈DNA結合。在PCR過程中，隨著擴增過程的進行，雙鏈PCR產物不斷增多，越來越多的熒光染料與雙鏈DNA結合。收集結合到雙鏈DNA上的SYBR
Green熒光信號就可以直接檢測各種PCR產物的量。我們用SYBR
Green熒光染料和位點特異PCR反應檢測HLAA2與非A2，說明這一類型的熒光PCR反應能在芯片上應用。
TaqMan探針熒光PCR反應與SYBR Green熒光染料PCR反應各有其優缺點。SYBR
Green熒光染料主要優點是不用針對每個PCR反應設計探針等，缺點是其特異性較差，原因是SYBR
Green是非選擇性的與雙鏈DNA結合。幸運的是通過測定解離曲線可以排除假陽性結果。用PCR芯片檢測腫瘤融合基因類型的工作中，如果無法設計數百個Taqman探針對不同的融合基因進行檢測，那么SYBR
Green熒光染料PCR反應則是很好的選擇。可以事先在芯片上點上檢測不同融合基因所需要的引物，然后將包括SYBR
Green熒光染料的反應液加入，檢測一個樣本多個不同融合基因的變化。
我們將芯片技術與熒光PCR技術相結合，利用TaqMan探針與SYBR
Green熒光染料檢測在微反應池內獲得的極其微量PCR產物。用一些臨床標本實例，觀察在基因芯片上進行不同的熒光摻入PCR反應，發現這些熒光PCR反應可以在芯片上順利進行，可以根據基因芯片上熒光的變化判斷基因的變異。為PCR基因芯片的臨床應用打下了基礎。
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2201-2204Experimental study of fluorescence PCR on PCR chips
(HAO Lin） ZHU Ping1）＊ YU Xiao-Mei2） ZHANG Da-Cheng） ZHAO Xin-Sheng OUYANG
Jian-Hua）
（1）Peking University First Hospital Beijing 100034；
（2）Department of
Microelectronics , Peking University Beijing 100871；
（3）College of Chemistry and Molecular Engineering , Peking University
Beijing 100871） Abstract Objectives: To prove if fluorescence PCR which
is used for different purposes can be carried through successfully on
PCR chips and whether the changes of fluorescence can be detected. To
compare the advantages and disadvantages of different methods of
fluorescence PCR. Methods: We designed and fabricated a PCR gene chip,
which is made of silicon wafer. A point mutation was identified by using
SSCP and sequencing a hereditary X chromosome linked sideroblastic
anemia family. A Taqman probe specifically to detect this point mutation
was designed and used in fluorescence PCR. We carried out the
fluorescence PCR on a gene chip and in tubes, and compared the results
from the two methods. We also used the 4 steps of site-specific PCR and
SYBR fluorescent staining to detect HLA-A2 on the chip and compared the
results with that from routine method. Results: A silicon PCR gene chip
was designed and fabricated which contains 192 reaction chambers. The
point mutation of G514A in the exon 5 of ALAS2 in a hereditary X-linked
sideroblastic anemia family was confirmed by PCR-SSCP and seqencing. The
results of the 6 members in the anemic family and 2 normal male
newborns using Taqman probe we designed and the fluorescence PCR on chip
were the same as those form routine method. The results of the routine
SYBR fluorescent staining PCR and the reactions on chip were consistent
with the expectations. Conclusions: Taqman fluorescence PCR can be
carried out successfully on the chip and is suitable for the detection
of point mutation and single nucleic polymorphorim. Fluorescence PCR
with SYBR fluorescence staining can be performed on the chip and may be
useful for the screening of fusion genes in cancer samples.
Keywords: PCR chip; Fluorescence PCR;Taqman probe;SYBR fluorescence staining；