核酸放大技术
核酸放大技术
杨朝勇
厦门大学化学化工学院
厦门大学分析科学重点实验室
福建省化学生物学重点实验室
固体表面物理化学国家重点实验室
联系电话: 86-592-2187601
Email: cyyang@xmu.edu.cn
PCR技术的创建
Khorana(1971)等提出在体外经DNA变性,与适当引物杂交,
再用DNA聚合酶延伸,克隆DNA的设想。
 1983年,Mullis发明了PCR技术,使Khorana的设想得到实现
1988年Saiki等将耐热DNA聚合酶（Taq）引入了PCR技术
1989年美国《Science》杂志列PCR 为十余项重大科学发明
之首,比喻1989年为PCR爆炸年
Mullis荣获1993年度诺贝尔化学奖。
三篇重要论文
The Unusual Origin of the Polymerase Chain Reaction
(Scientific American, 1990,262(4):56-61, 64-5 )
Primer-directed Enzymatic Amplification of DNA with
a Thermostable DNA Polymerase
(Science,1988,239(4839):487-91 )
Specific Synthesis of DNA In Vitro via a Polymerase
Catalyzed Chain Reaction (Methods in Enzymology,
1987,155:335-50 )
Mullis
的构思
1983年
引物
DNA聚合酶
DNA聚合酶
引物
特定DNA片段
94℃变性
50-65℃退火
XX℃延伸
94℃
55℃
37℃
1988年Saiki等将耐热DNA聚合酶（Taq）引入了PCR技术
Taq DNA聚合酶（thermus aquaticus)
100
(%)
酶
活
性
80
60
40
20
40 50
60 70 80 90 100
温度(℃)
94℃
55℃
PCR循环
72℃
PCR反应体系
Buffer
Taq
10~50mM Tris-HCl pH8.3
up to 50mM KCl or NaCl
1.5mM or higher MgCl2
1.5~3u
dNTP
Primer F/R
50~200uM each dNTP
0.2~1uM each primer
人基因组DNA 0.1ug～1ug
Template DNA
DD water
大肠杆菌
10ng～100ng
DNA
0.5ng～5ng
质粒DNA
0.1ng～10ng
补全
25uL or 50uL
Gelatin(明胶) or BSA
100ug/ml
Non-ionic detergents such as Tween-20 or Triton X-100
影响PCR的因素
1. Denaturing temperature and time
Normally: 1min at 94℃
对于较短的模板，变性时间可以减少为30s或者更
短。也可以增加变性温度，减少时间，比如95℃
30s，96℃ 15s。
2. Annealing Temperature and Time
Tm=4(G+C)+2(A+T) ℃
Annealing temperaure(Ta)=the loweat Tm of the pair of primers - 5℃
Annealing time normally：30s or less
Ta太低，碱基错配和部分退火都容易发生，所以容易引起引物与非模板DNA
的非特异性结合，继而进行非特异性扩增，使得目标产率降低
Ta太高，太接近Tm，引物会很难退火到模板DNA上，使得产率降低
3. Elongation Temperature and Time
Normally: 70~72℃, 0.5~3min
70℃
100b/s
2kb/min
3kb/3min
……
Longer time in later cycles
引物设计规则
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
长度为17～28mer,太短易形成错配降低特异性，而太长也会降低特异性，
并且降低产量
50～60％(G+C)，且上下游引物序列GC含量的差异不要太大
Tm在55～80℃,且两条引物的Tm应接近
3‘端以G or C，or CG or GC结尾，可使引物与模板稳定结合
3’端避免连续3个或更多C or G，因为G or C有很强的退火稳定性，这样容
易与富含GC的序列错配
两个引物的3‘端应避免互补，否则会先于和模板配对而形成引物二聚体
避免引物自身内部互补形成发夹结构
DNA双链形成所需的自由能G ，应该以5’端向 3’端递减，3’端G最好不
要高于9.0 kcal/mol；  G高于4.5 kcal/mol时易引发上述两种结构的产生
引物所在的模板区域应该位于外显子区，最好跨越一个内含子区，这样便
于对扩增出来的片段进行功能鉴定和表型分析
5’端对PCR影响不太大，可以引进修饰位点和标记物
Variants of PCR
 Quantitative PCR (or Q-PCR)
 Asymmetric PCR
 Multiplex-PCR
 Nested PCR
 Hot-start PCR
 Touchdown PCR
 Digital PCR
RT-PCR (Reverse Transcription PCR )
…
Real-Time PCR
Currently available chemistries for fluorescence detection
Nonspecific detection
Specific detection
SYBRTM Green
TaqManTM
SYBRTM Gold
Hybridization Probes
YoYoTM-1
Molecular Beacons
Yo-ProTM-1
ScorpionsTM
AmplifluorTM
Lanthanide
Quencher-labelled primers I
ResonSenseTM
Quencher-labelled primers II
AnglerTM
LuXTM primers
HybeaconsTM
Cationic conjugated PNA
Light-upTM
EclipseTM
Ds complex probes
CycliconsTM
Nanoparticles
SYBR Green®
SYBR Green® is a fluorescent dye that binds to double stranded
DNA with great specificity.
It is a synthetic asymmetrical cyanine dye, which was introduced in
the early 1990s by Molecular Probes®.
SYBR Green® is very sensitive; it is 25 times more sensitive than
ethidium bromide, another commonly used dye for visualizing DNA .
It preferentially binds double stranded DNA, but it can also bind
single stranded DNA with reduced fluorescence
SYBR Green Chemistry
TaqMan® Probe
TaqMan probes are hydrolysis probes developed by Applied
Biosystems to increase the specificity of real-time PCR assays.
 TaqMan probes consist of a fluorophore covalently attached to the
5’-end of the oligonucleotide probe and a quencher at the 3’-end.
The TaqMan probe principle relies on the 5´–3´ nuclease activity of
Taq polymerase to cleave a dual-labelled probe during hybridization
to the complementary target sequence and fluorophore-based
detection.
TaqMan Chemistry
Scorpion® Primer
Scorpion technique was developed by Dr. David Whitcombe of DxS
Ltd. Scorpion primers are bi-functional molecules in which a primer is
covalently linked to the probe. The molecules also contain a
fluorophore and a quencher.
In the absence of the target, the quencher nearly absorbs the
fluorescence emitted by the fluorophore. During the Scorpion PCR
reaction, in the presence of the target, the fluorophore and the
quencher separate which leads to an increase in the fluorescence
emitted. The fluorescence can be detected and measured in the reaction
tube.
Scorpion Primer
Molecular Beacons
Molecular beacons are single stranded hairpin shaped oligonucleotide probes. In the
presence of the target sequence, they unfold, bind and fluoresce.
A molecular beacon consists of 4 parts, namely
• Loop: This is the 18-30 base pair region of the molecular beacon which is
complementary to the target sequence.
• Stem: The beacon stem sequence lies on both the ends of the loop. It is
typically 5-7 bp long at the sequences at both the ends are
complementary to each other.
• 5' fluorophore: Towards the 3' end of the molecular beacon, is attached a dye
that fluoresces in presence of a complementary target.
• 3' quencher : The quencher dye is covalently attached to the 3' end of the
molecular beacon and when the beacon is in closed loop shape,
prevents the fluorophore from emitting light.
Beacon Chemistry
Target
Molecular beacon
FRET Probes
 FRET probes are a pair of fluorescent probes placed in close proximity.
Fluorophores are so chosen that the emission spectrum of one overlaps significantly
with the excitation spectrum of the other.
 During FRET, the donor fluorophore excited by a light source, transfers its energy
to an acceptor fluorophore when positioned in the direct vicinity of the former.
 The acceptor fluorophore emits light of a longer wavelength, which is detected in
specific channels. The light source cannot excite the acceptor dye.
FRET Chemistry
2
1
3
Reverse Transcriptase PCR
Asymmetric PCR
 Asymmetric PCR is used to preferentially amplify one strand of the original DNA
more than the other. It finds use in some types of sequencing and hybridization
probing where having only one of the two complementary stands is ideal. PCR is
carried out as usual, but with a great excess of the primers for the chosen strand.
Linear-After-The-Exponential (LATE)-PCR
Nested PCR
• Nested PCR refers to a pair of PCRs run in
series each with a pair of primers flanking
the same sequence.
• The first PCR amplifies a sequence as seen
in any PCR experiment. The second pair of
primers (nested primers) for the second PCR
bind within the first PCR product and
produce a second PCR product that is
shorter than the first one.
• The technique, because it uses four specific
primers, rather than two, has greater
specificity than regular PCR. It can also
yield detectable product in cases where
simple PCR fails to do so.
Hot Start PCR
Touchdown PCR
♫Touchdown PCR is another modification of conventional PCR that
may result in a reduction of nonspecific amplification.
♫It involves the use of an annealing temperature that is higher than the
target optimum in early PCR cycles. The annealing temperature is
decreased by 1°C every cycle or every second cycle until a specified or
'touchdown' annealing temperature is reached.
♫The touchdown temperature is then used for the remaining number of
cycles. This allows for the enrichment of the correct product over any
non-specific product.
1010101010101010101110101010101010101011
0101011010110101011011011010101000100101
1101011010101010101010100101010101001010
101010100010101010101001010100101010010
100101001010010010101001010100101001010
100101010010101010010100101100101010010
1001001010101010101110010101010101010110
1010101101001010101010111111011001001010
0101001001010101110101011010100101000000
00011010101101111111111101010101010101010
101010101010010101010101010101010101010
010101010101010101001010101001010101001
01010101010101010100000000111111111101010
1101001110101010101010101010101100101100
0111000100001010101000000111000011110100
00111100101010101011011001011011110101001
11100110110010110100100011001101110101001
0010010101010101011100101010101010101101
0101011010010101010101111110110010010100
1010010010101011101010110101001010000000
00110101011011111111111010101010101010101
010101010100101010101010010100101010010
101001010101001010
Digital PCR
What is Digital PCR
Digital PCR concept was mentioned for the first time in Nucleic
Acids Res. 1997, Vol 25, No 10,1999-2004 by Kalinina et al
What is Digital PCR
“Digital PCR” was published for the first time in PNAS. 1999, Vol
96, 9236 by B. Vogelstein et al
Schematic of Dig-PCR
Vogelstein B, Kinzler K W PNAS 1999;96:9236-9241
©1999 by The National Academy of Sciences
Digital PCR
The technique is to amplify a single DNA
template from minimally diluted samples,
therefore generating amplicons that are
exclusively derived from one template and
can be detected with different fluorophores
or sequencing to discriminate different
alleles.
Assembly PCR
Assembly PCR provides a method for the
synthesis of long DNA sequences from large
numbers of oligonucleotides.
Rolling circle amplification
Rolling circle amplification (RCA) technology, a proprietary amplification process
developed by Molecular Staging Inc, has significant advantages in terms of sensitivity,
multiplexing, dynamic range and scalability. The steps of this procedure are:
1. A short DNA probe anneals to a target DNA of interest, the probe then acts as a primer for
a Rolling Circle Amplification reaction.
2. The free end of the probe anneals to a small circular DNA template. A DNA polymerase is
added to extend the primer.
3. The DNA polymerase extends the primer continuously around the circular DNA template
generating a long DNA product that consists of many repeated copies of the circle.
4. By the end of the reaction, the polymerase generates many thousands of copies of the
circular template, with the chain of copies tethered to the original target DNA. This allows
for spatial resolution of target and rapid amplification of the signal. The use of forward and
reverse primers can change the above linear amplification reaction into an exponential mode
that can generate up to 1012 copies in 1 hour.
Rolling circle amplification
Rolling circle amplification
Helicase-Dependent Amplification
• Helicase-dependent amplification (HDA) is a method for in vitro
DNA amplification like PCR, but that works at constant temperature.
• Helicase are involved in all biological processes.
-DNA replication
-DNA repair and recombination
-Transciption
-Translation
-RNA splicing
Nature uses an enzyme called helicase to
separate DNA during replication
Arthur Kornberg and Tania Baker, DNA Replication, 1992
Helicase Dependent Amplification (HDA)
Helicase separates
DNA duplex
Primers binds
to ssDNA
repeat
Helicase
Primer
Polymerase
Polymerase extends the
primer, making a new strand
Amplification of target sequences from bacterial genomic
DNA without prior denaturation
M 1 2
300-bp
200-bp
100-bp
97bp
Lane 1: Two-step HDA, 95°C 3 min and then 37°C 60 min.
Lane 2: One-step HDA, 37°C 60 min from beginning to end.
One Tube,
One Temperature,
From the beginning to the end
The simplest amplification scheme
J. Clin.Microbiol, 2008, 46, 1534-1536