邻位连接技术(PLA)
2011-11-23 04:36阅读：
在PLA技术中，不同类型的抗体上，连接有不同的核酸适体（1），另外二条寡核苷酸探针（2），各自都能与二个不同的核酸适体互补配对，在连接酶的作用下，二条寡核苷酸探针产生连接，因此再通过PCR扩增，没有读出DNA序列的要求，只是通过滚环复制产生“多连体”，再有荧光标记的单链核苷酸（3）与新合成的DNA序列配对，引入荧光标记，并且结合点众多，放大了靶点得荧光信号。不需要序列信息，只要有PCR扩增超长长度的产物就行。
换句话说，有无蛋白质相互作用，就看有无DNA连接反应发生。
二条寡核苷酸探针（2），各自都能与二个不同的核酸适体互补配对
DNA连接，之后是DNA复制（PCR反应）
通过滚环复制产生“多连体”
荧光标记的单链核苷酸（3）与新合成的DNA序列配对
大量的荧光标记杂交在一个靶点上
we decided to monitor and quantify the
Dnmt1/PCNA interaction by proximity ligation
in situ assay
(P-LISA) in cells . As illustrated by the Figure
1A,
the principle of this assay is based on
the staining of
Dnmt1 and PCNA proteins by two antibodies, which are next
revealed by
secondary antibodies conjugated with
oligonucleotides. In presence of hybridization solution
and ligase, the two oligonucleotides form with PLA a circle in case
of close proximity of proteins i.e. Dnmt1 and PCNA, here. Then,
polymerase and nucleotides participate to the formation of the
rolling circle amplification,
which are visualized in red
fluorescence.
A
B
Figure 1. Detection of
endogenous Dnmt1/PCNA interactions using P-LISA.
(A) Schematic representation of the Dnmt1/PCNA proximity ligation
in situ assay (P-LISA).
(B) Calibration of the microscopy (Axiovert 200 M Zeiss, Le Pecq,
France) with ApoTome module. Left: picture of calibration performed
with calibration balls (4
μm, Vue X-Y, Molecular
Probes F36909). Right: picture of calibration performed with
calibration balls (2.5
μm, Vue X-Z, Molecular Probes
F36909). Blue and red were merged in lateral and axial.
(C) Decovolving of picture realized with calibration balls (140
nm).
(D) Dnmt1/PCNA interactions were visualized in MCF10A cells.
Red dots symbolize Dnmt1/PCNA interactions.
Nucleus/DNA are stained in
blue via the use of
DAPI. Top left: ApoTome view, top right: orthogonal view,
bottom: 3D views.
引自：
Proximity ligation in situ assay for monitoring
the global DNA methylation in cells.BMC
Biotechnology
Volume
11 2011.Eric
Hervouet1,2 ,
Philippe
Hulin2,3 ,
François M
Vallette1,2 and
Pierre-François
Cartron1,2
PLA is an antibody-based method in which either a single or two
proteins (or antigens) are immunolabeled first with two primary
antibodies and then with different species-specific secondary
antibodies conjugated to complementary oligonucleotides (
8,
9).
When two antibody molecules are in close proximity, the
complementary DNA strands can be ligated, amplified, and visualized
with a fluorescent probe as distinct puncta (Figure 1). Each spot
may represent a single complex containing each of two interacting
proteins (or antigens).
The maximal distance between the
secondary antibodies in this assay is ~16 nm, only
slightly larger than that for resonance energy transfer between
fluorophores (~10 nm), the most common approach used to infer GPCR
oligomerization. By measuring close proximity, PLA allows a
validation in vivo of the molecular proximity of two endogenous
proteins, something that cannot be established with simple
colocalization studies, thereby making it possible to interrogate
the existence and localization of interactions in vivo.
Figure 2. The Duolink assay principle. (A)
Two primary antibodies raised in different species are used to
detect two target antigens of interest [here as examples,
gray/green (left) against a target protein (green), and light
gray/yellow (right) against a second target protein (yellow)]. (B)
Each species-specific secondary antibody (dark gray and light gray,
respectively) provided in the Duolink kit has a unique short DNA
strand attached to it (black line). When the secondary antibodies
are in close proximity, the DNA strands can interact through a
subsequent addition of two other circle-forming DNA
oligonucleotides (circular black line). The distance between the
two secondary antibodies is a maximum of 16 nm as calculated from
the number of nucleotides in the attached DNA arms. (C) After
enzymatic ligation, the two added oligonucleotides are amplified
via rolling circle amplification using a polymerase to yield a long
concatemeric copy of the circle formed by ligation. (D) After the
amplification reaction, labeled complementary oligonucleotide
probes are added to highlight the product (red circles).
引自：Detection of antigen interactions ex vivo by proximity ligation
assay: endogenous dopamine D2-adenosine A2A receptor complexes in
the striatum.BioTechniques.2011.Pierre Trifilieff
1,2, 3, Marie-Laure Rives
3,4,
5, 6, Eneko Urizar*
4,5,
6, Rebecca A. Piskorowski
1, Harshad D. Vishwasrao
1, John Castrillon
3, Claudia Schmauss
2,5, Maria Slättman
7, Mats Gullberg
7, and Jonathan A. Javitch
4,5,
6, 8
转帖：
蛋白质检测新技术——邻位连接技术及初步应用
唐
娜
1
沈志强
1，2 管
宇
1 王金良
1
(1山东省滨州畜牧兽医研究院预防兽医学与动物生物技术重点实验室 滨州256600)
(2山东绿都生物科技有限公司 滨州256600)
邻位连接技术是近几年建立的一种可以用于研究蛋白质的定量、定位、相互作用、修饰以及功能的新DNA连接技术。该技术通过一对邻位探针将特异性蛋白质检测转换为DNA序列分析，可以直接分析复杂的生物样品，检测灵敏度和精确度高。本文总结了国外相关报道，对PLA技术原理，PLA技术的种类进行了系统的介绍，并介绍了该方法在生物标记因子检测、蛋白质相互作用、蛋白质一DNA相互作用以及疾病诊断中的初步应用。
1
PLA技术原理
1．1 PLA技术原理
DNA连接技术早在20世纪70年代首次发现连接酶之后便被应用于实验分析中，最早用于研究tRNA的分子作用机制。此后陆续出现了多种新的连接技术，如用于单核苷酸多态性标记(single
nucleotide polymorphisms，SNPs)的寡核苷酸连接分析(oligonucleotide ligation
assay，OLA)技术，以及用于基因组和蛋白质组分析的Padlock探针技术，这种寡核苷酸可以和特定的核酸序列反应环化而进行滚环扩增(rolling
circle replication，RCA)，而PLA便是应用一种类似机制的探针讲行蛋白后检测的技术。
PLA技术检测蛋白质的核心是一对邻位探针。该对探针将单链DNA与一对蛋白识别因子相连接，使其可以通过免疫吸附或其他方法同步结合到一个待测蛋白分子上。PLA反应分为3步：1)样品与带有靶特异性抗体的一对邻位探针共孵育，2条邻位探针双双与靶相应位点结合；2)加入连接试剂和PCR扩增试剂，在连接酶作用下使得同一目的蛋白上的邻位探针对DNA尾部的游离5’端和3’端与
