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PCR:聚合酶链式反应

PCR: Polymerase Chain Reaction

你好,欢迎来到这个关于聚合酶链式的视频。
Hello, and welcome to this video about polymerase chain
这是一个反应,通常被称为PCR。
reaction, or as it’s more commonly known, PCR.
DNA,正如我们所知,是所有生命的蓝图。
DNA, as we know it, is the blueprint for all of life.
PCR是我们利用一段特定的DNA。
PCR is how we can take a specific piece of DNA
通过一个过程复制很多,很多的拷贝。
and put it through a process to make many, many copies of it.
这是非常重要的,因为它允许
This is very important, as it lets
我们迅速做出足够的DNA,这样我们可以
us make enough DNA quickly so that we can actually
运行实验。
run experiments.
整个过程有很多共同点
The whole process has a lot in common with how
的复制机器复制特定的一个模板
a copy machine makes copies of a certain piece of paper.
聚合酶链反应发生,我们首先需要混合
To make PCR happen, we need to first mix
所有的材料一起加入在一个小塑料管里
all of the ingredients together in a small plastic tube, which
就是我们看到的左边。
is what we’re looking at here on the left.
接下来,我们要放大这个管子的内部。
So we’re going to zoom in on this tube
看看每个成分。
and take a look at each ingredient.
我们首先需要的是一个缓冲液。
The first thing we need is a buffer.
缓冲液是主要是水,内部含有一些离子。
So the buffer is just mostly water with some ions in it.
这是旨在使pH值恒定
It’s designed to keep the pH constant so
其余的成分可以做实际工作。
that the rest of the ingredients can do their actual jobs.
接下来我们需要添加核苷酸到混合物中。
The next thing we’d need to add into the mix
这样我们可以使用他们制造DNA。
is the nucleotides so that we can use them to make DNA.
可以认为这与复印过程中使用纸和墨水
The way to think of this is like the paper and ink
是类似的。
of a copy machine.
接下来原材料。
It’s the raw material.
记住,我们有四个DNA碱基,腺嘌呤、胸腺嘧啶,
Remember that we have four DNA bases, adenine, thymine,
胞嘧啶、鸟嘌呤。
cytosine, and guanine.
也要记住,腺嘌呤
Also be sure to remember that adenine always
对胸腺嘧啶、鸟嘌呤和胞嘧啶总是配对的。
pairs with thymine, and cytosine always pairs with guanine.
我们就是用这一特点
This is how we’re going to use this in a little while
来制造我们的DNA拷贝。
to actually make our copies of DNA.
接下来我们需要在混合物中添加
The next thing we want to add into our mix
DNA模板。
is the DNA template.
考虑原始拷贝。
The way to think about this is that this is the original copy
当你进行拷贝之前。
that you would put into a copy machine.
记住,DNA是有方向的,
Remember that DNA has a direction, which
我们所说的5 ‘至 3 ‘,并且链是反向平行。
we call 5′ to 3′, and that the strands are anti-parallel so
总是从5 ‘端开始碱基匹配,3 ‘端结束。
that the 5′ end always matches up with the 3′ end
其他的链,反之亦然。
of the other strand and vice versa.
接下来,我们需要加入PCR引物。
The next piece that we need to add in are the PCR primers.
这些引物是小的DNA片段,
These primers are small pieces of DNA,
它们是我们想要的DNA拷贝的末端。
which mark the end of the segment of DNA we want to copy.
它讲述了反应从哪里开始和停止的地方。
It tells the reaction where to start and where to stop.
接下来,我们说一下放大
So let’s say that we want to amplify
整个小DNA片段
this entire small piece of DNA that we
在我们的反应中。
have here in our reaction.
我们需要一个短的引物,
Then we will need one short primer,
附加到DNA的两端。
which will attach to each end of the DNA
导向正确的方向。
and be oriented in the correct direction.
在我们的反应中添加最后一样物质
The last thing to add into our reaction
被称为聚合酶。
is called the polymerase.
聚合酶就像复印机。
The polymerase is like the copier itself.
这台机器通常会
It is the machine that will actually
使用DNA碱基,DNA模板和引物
use the DNA bases, the template DNA, and the primers
复制的DNA链。
to make copies of the DNA strand.
现在,我们准备设置我们的反应。
Now we’re ready to set up our reaction.
在这里,我们的反应都在管里
Here we have our reaction in the tube,
然后,打开机器。
and we’re now putting it in this machine, which
它被称为 热循环仪。
is called a thermocycler.
所有的热循环仪,都需要设置
All the thermocycler does is change
管子温度,然后设定时间。
the temperature of the tube after a set amount of time.
这个温度的设定
It’s this temperature change that’s
要允许聚合酶来完成工作。
going to allow the polymerase to do its work.
这里,我们返回管中。
So here we are, back in the tube.
你可以看到我们所有的组件
And you can see all of our components
从顶部的标签之前,除了,
from before labeled at the top, except for,
当然,模板DNA链,这是底部。
of course, the template DNA strand, which is at the bottom.
在这种情况下的我们做了一段时间的模板
I’ve made the template a bit longer in this case
所以我们可以真正看到每一步发生。
so we can really see each step occurring.
记得我们有所有这些组件的许多拷贝。
Remember we have multiple copies of all of these components,
不是每个拷贝,都能停止我们
not just the one copy of each I’ve stuck up
顶部是一个说明。
at the top as a legend.
您还会注意到,温度
You’ll also notice that the temperature
在左上角。
is in the upper left corner.
现在,在18度
And right now, it’s at 18 degrees
摄氏度,在室温下是正确的。
Celsius, which is right around room temperature.
这些就是我们打开热循环仪开始前的事情。
This is where things start when we turn the thermocycler on.
现在我们要进入第一步,
Now we’re going to enter the first step, which
被称为融化。
is called melting.
我们已经提高了温度到95摄氏度。
We’ve now raised the temperature to 95 degrees
略低于水的沸点。
C, which is just under the boiling point for water.
这是断开氢键,
This breaks apart the hydrogen bonds,
使原有来在一起的DNA链
which hold the DNA strand together,
给我们两个单独的互补的DNA链。
to give us two separate complementary strands of DNA.
我们现在降低温度的退火阶段。
We now lower the temperature for the annealing phase.
在这种情况下,我选择了55度,
In this case, I’ve picked 55 degrees,
利用它你可以随意放大DNA数量。
but it can vary depending on the DNA you’re trying to amplify.
这被称为退火步骤
This is called the annealing step
由于引物现在可以进来和绑定,或退火,
because the primers can now come in and bind, or anneal,
在这个地方补充合成单链
to the complementary single strand at the place
在那里他们匹配,如下所示。
where they match, as shown here.
他们现在可以被复制。
They are now ready to be copied.
我们现在进入最后一步,延伸。
We now enter the final step, elongation.
在这一步中,我们提高温度。
In this step, we raise the temperature
到72度,它允许聚合酶
to 72 degrees, which allows the polymerase
识别引物绑定到单独的DNA链。
to recognize the primers bound to the single strand of DNA.
聚合酶结合,开始向下游运动
The polymerase binds and begins moving down
现有的DNA链,总是这样
the existing strand of DNA, always
添加互补碱基至3 ‘末端
adding the complementary base to the 3′ end
不断延长的DNA链。
of the growing DNA strand.
当聚合酶到达
So as the polymerase comes off as it reaches
链的末尾,我们现在拥有了
the end of the strand, we now have
两个完整的双链DNA分子。
two complete double-stranded DNA molecules
从一个双链DNA
from what was just one double-stranded DNA
分子开始。
molecule before.
所以这个过程可以重复一遍又一遍。
So this process can be repeated over and over again.
这里我们缩小,现在每一行代表
Here we’ve zoomed out, with each line now representing
一个DNA链。
a single strand of DNA.
当我们经过多个周期,
As we go through the multiple cycles,
我们增加DNA分子的总数
we increase the total number of DNA molecules
每一轮提高两倍。
by a factor of two each round.
每轮,我们得到了越来越多的DNA复制
So with every round, we get more and more DNA copies
我们的目标。
of our target.
这就是PCR如何复制DNA
This is how PCR generates copies of a piece of DNA
研究人员在他们的实验中使用。
for researchers to use in their experiments.
这是基本的PCR是如何工作的。
And that’s the basics of how PCR works.
感谢收听
Thanks for listening.
[奏乐]
[MUSIC PLAYING]

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