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怎样才能去掉黄褐斑PCR常见问题的精辟总结

作者:陕西保健网
来源:http://www.xapfxb.com/yuer
更新日期:2021-02-01 23:29

-红斑狼疮怎么治疗

2021年2月1日发(作者:胃穿孔手术)
PCR
的优越性与局限性

时间
:2010-02-27 14:56:24
来源
:
作者
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聚合酶链反应即
PCR
技术,
因为其 对基因或特定核酸序列在短时间内的极大的扩增效率,

在感染性疾病、肿瘤、遗传病、寄生虫 病、法医学、动植物和考古等的诊断和研究中得到了
广泛的应用,并在某些方面几乎是难以替代的。但是 ,像自然界的任何事物一样,
PCR
也有
其局限性,稍不注意,就很容易造成错误的判 断。

感染性疾病由病原微生物引起,主要有病毒、细菌、衣原体、支原体和螺旋体等,在PCR
出现以前,
这些病原体通常采用培养、
免疫学方法测定特异抗原抗体,核酸杂交等进行临床
检测,
但这些方法对某些病原体要么难以进行
(如导致结核病 的结核杆菌、
引起性病的沙眼
衣原体和诱发肝炎的乙肝丙肝病毒等的培养)

要么难以判断体内的复制情况或检测的灵敏
度不够(如乙肝丙肝病毒和人免疫缺陷病毒等的抗原抗体检测 及核酸杂交检测等)。
PCR

出现,
可以说从根本上改变了这一点,
其极高的检测灵敏度和特异性,
使难培养病原体的检
测变得迅捷而又准确。
各种定量
PCR
模式的出现,
又使其抗病毒疗效的判断可以很客观地从
病毒核酸的量上 反映出来。并且,
PCR
用于病原体感染的血液筛查,可以检出抗原抗体尚未
出现的“ 窗口期”内的感染,从而避免经输血引发的感染性疾病的传播。

遗传基因的异常可致遗传病的 发生,人类遗传病约有
3000
多种,患者占总人口数的
10
%。
遗 传病一般可分为单基因、
多基因及染色体遗传病。
这些遗传病通常是由于基因突变、
基 因
缺失、
染色体错位所致。
以前常用的诊断方法有家系谱分析、
染色体检查< br>(特别是显带法)

生物化分析等。

PCR
技术由于其对基 因体外扩增快速灵敏的特点,
结合其他多种分子生物
学手段可以检出大部分已知的基因突变、< br>基因缺失、
染色体错位等,
应可成为遗传病诊断的
最有效、最可靠的方法之一。

法医学也因为
PCR
技术的出现,
进入到了分子物证的时代,并可以精确到个体确认水平,

在分子水平上对血斑、精斑、毛发、
组织碎片乃至 微量残留细胞等法医物证的确认,远较以
前的血清学方法确实可靠,
如以前的血型鉴定,
只能是排除不同血型者,
而无法做到个体确
认。并且
PCR
因为其极大的扩 增效率,可以对极少量物证,如一根毛发、一滴血液、极小精
斑、体液中的脱落细胞等进行分析,
从而确定个体。可以说,
当代法医鉴定在个体鉴别上已
离不开
PCR
技术。

众所周知,
肿瘤与遗传有关,除已知的单基因遗传肿瘤,
如视网膜细胞瘤、
肾母细胞瘤等以
外,
几乎所有的肿瘤细胞中都可以观察到原癌基因的重排,
这 些基因的变化常导致细胞增殖
与凋亡失控,最终形成肿瘤。
PCR
扩增检测技术,使癌 基因和抑癌基因及其状态的检测,变
得简便、快捷而又容易。此外,使用反转录
PCR
检测特定肿瘤抗原的
mRNA
,很容易监测到
癌转移的发生。

PCR
这种基因扩增技术因其简单易行,应用现已极为广泛,但其影响因素很多,如标本或核< br>酸纯化过程中出现的扩增反应抑制物、扩增仪孔间温度的差异以及核酸提取中的随机误差,
可造成 假阴性结果的出现。
又如,
以前扩增产物的污染和核酸提取中标本间的交叉污染,
也< br>很容易出现假阳性结果。因此,
PCR
临床应用必须要有严格的实验室分区、实验室管理 和质
量控制措施,
只有在充分了解
PCR
测定的不确定性的基础上,
采取相应质控措施,
才能确保
实验结果的准确性,而不致出现重大判断失误。因为
PC R
扩增,其得到极大的检测灵敏度,
但同时其对检测中的错误也有极大的放大作用。因此,对于
PCR
检测结果的报告必须慎重,
尤其是在该结果会产生重大后果的时候,
如 重大感染性疾病、
遗传病、
法医物证鉴定、
亲子
鉴定等,必须在有相应严格质 控措施(如内质控、阴性和阳性质控)的情况下重复测定,才
可以报告结果,并做出实事求是的解释。< br>
PCR
产物量小或没有目的产物

时间
:2010-02-27 14:33:26
来源
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作者
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Possible Causes: Components

· Primer concentration is too low.

· Primer concentrations not balanced. Make sure
primers are present in equal
concentrations.
· New primers are required. Some primers are immune to optimization. See Primer
Design.
· Nested primers are required. Reamplify dilutions (1:10 to 1:1000) of the first
reaction using nested primers .
· Contamin
ated primer. See Rescuing Contaminated PCR
Primers and Wayward PCR Primers.

· Template concentration is too low. Use a higher concentration of template.

·
Template
concentration
is
too
high.
Excessive
template
can
inhibit
the
reaction
by binding all the primers .
·
Template
is
degraded.
Check
the
integrity
of
the
template
by
electrophoresis
after
incubation.
·
Template:
Target
sequence
is
not
present
in
target
DNA.
Redesign
your
experiment
or try other sources of target DNA.
· Mg++ concentration is too lo
w. This may compromise enzyme activity so try
increasing the concentration by 0.2 mM
increments.
· dNTP
concentration is too low. Normally concentrations between 20
- 200
μ
M are
optimal.
· dNTPs degraded. Keep nucleotides frozen in aliquots, thaw quickly a
nd keep on
ice once thawed. Avoid multiple
freeze/thaw cycles.

Troubleshooting for PCR and multiplex PCR

Troubleshooting discussion is based on the PCR protocol as described in the table
below. All reactions are run for 30 cycles.
COMPONENT VOLUME FINAL CONCENTRATION

aved ultra-
filtered water (pH 7.0) 20.7?L
-
2.10x PCR Buffer* 2.5?L 1x

mix (25 mM each nucleotide) 0.2?L 200 ?M (each nucleotide)

mix (25 pmoles/?L each primer) 0.4?L 0.4 ?M (each primer)

DNA polymerase (native e
nzyme) 0.2?L 1 Unit/25 ?L

c DNA template (100 ng/?L) 1.0?L 100 ng/25 ?L

*
The
10x
PCR
buffer
contains:
500
mMKCl;
100
mMTris-HCl
(pH
8.3);
15
mM
MgCl2
(the
final
concentrations
of
these
ingredients
in
the
PCR
mix
are:
50
mMKCl;
10
mMTris-HCl;
1.5 mM MgCl2).
QUESTIONS SOLUTIONS

1. I get (many) longer unspecific products. What can I do?

Decrease annealing time
Increase annealing temperature
Decrease extension time
Decrease extension temperature to 62-
68? C

Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at
1.5-2mM.
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
Take less primer
Take less DNA template
Take less Taq polymerase
If none
of the above works: check the primer for repetitive
sequences (BLAST align
the sequence with the databases) and change the primer(s)
Combine some/all of the above
2. I get (many) shorter unspecific products. What can I do?

Increase annealing temperature
Increase annealing time
Increase extension time
Increase extension temperature to 74-
78? C

Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at
1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant
Take less primer
Take less DNA template
Take less Taq polymerase
If none
of the above works: check the primer for repetitive
sequences (BLAST align
the sequence with the databases) and change the primer(s)
Combine some/all of the above
3. Reaction was working before, but now I can't get any product. Make sure all
PCR ingredients are taken in the reaction (buffer, template, Taq, etc)

Change the dNTP solution (very sensitive to cycles of thawing and freezing,
especially in multiplex PCR)
If
you
just
bought
new
primers,
check
for
their
reliability
(bad
primer
synthesis
?)
Increase primer amount
Increase template amount
Decrease annealing temperature by 6-
10? C and check if you get any product. If you
don't,
check
all
your
PCR
ingredients.
If
you
do
get
products
(including
unspecific
ones) reaction conditions as described above.
Combine some/all of the above
4.
My
PCR
product
is
weak.
Is
there
a
way
to
increase
the
yield? Gradually
decrease
the annealing temperature to the lowest possible.

Increase the amount of PCR primer
Increase the amount of DNA template
Increase the amount of Taq polymerase
Change
buffer
(KCl)
concentration
(higher
if
product
is
lower
than
1000bp
or
lower
if product is higher than 1000bp)
Add adjuvants. Best, use BSA (0.
1 to 0.8 ?g/?L final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol.
Check primer sequences for mismatches and/or increase the primer length by 5
nucleotides
Combine some/all of the above
5.
My
two
primers
have
very
different
melting
temperatures
(Tm)
but
I
cannot
change
their locus. What can I do to improve PCR amplification?

An easy solution is to increase the length of the primer with low Tm. If you need
to keep the size of the product constant, add a few bases at the 3' end. If size
is not a concern, add a few bases at either the 3' or the 5' end of that primer.
6.
I
have
a
number
of
primer
pairs
I
would
like
to
use
together.
Can
I
run
a
multiplex
PCR with them?. How?

Very likely, yes.
Try amplify all loci seaprately using the same PCR program. If one of the primer
pairs yields unspecific products, keep the cycling conditions constant and
change other parameters as mentioned above (#1 and #2).
Mix equimolar amounts of primers and
run the multiplex
reaction
either in the same
cycling conditions or by decreasing only the annealing temperature by 4?C.

If some of the loci are weak or not amplified, read below !!
7. How many loci can I amplify in multiplex PCR at the same time?

Difficult to say. The author has routinely amplified from 2 to 14 loci.
Literature describes up to 25 loci or so.
8. One or a few loci in my multiplex reaction are very weak or invisible. How can
amplify them?

The first choice should be increasing the amount of primer for the
the same time with decreasing the amount of primer for all loci that can be
amplified. The balance between these amounts is more important than the absolute
values used !!.
Check primer sequences for primer-primer interactions
9. Short PCR products in my multiplex reaction are weak. How can I improve their
yield?

Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at
1.5-2mM
Decrease denaturing time
Decrease annealing time and temperature
Decrease extension time and temperature
Increase amount
of primers for the
loci
while decreasing
the amount for the

Add adjuvants. Best, use BSA (0.1 to 0.8 ?g/?L final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol
Combine some/all of the above
10.
Longer
PCR
products
in
my multiplex
reaction
are weak.
How
can
I
improve
their
yield?

Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at
1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
Increase denaturing time
Increse annealing time
Decrease annealing temperature
Increase extension time and temperature
Increase amount
of primers for the
loci
while decreasing
the amount for the

Add adjuvants. Best, use BSA (0.1 to 0.8 ?g/?L
final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol
Combine some/all of the above
11. All products in my multiplex reaction are weak. How can I improve the yield?

Decrease annealing time in small steps (2? C)

Decrease extension temperature to 62-
68? C

Increase extension time
Increase template concentration
Increase overall primer concentration
Adjust Taq polymerase concentration
Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
Add adjuvants. Best, use BSA (0.1 to 0.8 ?g/?L final concentration). You can also
try 5% (v/v, final concentration) DMSO or glycerol
Combine some/all of the above
12. Unspecificproducts appear in my multiplex reaction. Can I get rid of them
somehow?

If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at
1.5-2mM
If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration
at 1.5-2mM
Gradually increase the annealing temperature
Decrease amount of template
Decrease amount of primer
Decrease amount of enzyme

-红斑狼疮怎么治疗


-红斑狼疮怎么治疗


-红斑狼疮怎么治疗


-红斑狼疮怎么治疗


-红斑狼疮怎么治疗


-红斑狼疮怎么治疗


-红斑狼疮怎么治疗


-红斑狼疮怎么治疗



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