PCR: Polymerase Chain Reaction - Free Educational videos for Students in K-12

PCR: Polymerase Chain Reaction - By MITK12Videos

Transcript


00:06	Hello and welcome to this video about polymerase chain reaction
00:09	or as it's more commonly known PcR . D .
00:13	N . A . As we know it is the
00:14	blueprint for all of life . PcR is how we
00:17	could take a specific piece of D . N .
00:18	A . And put it through a process to make
00:21	many , many copies of it . This is very
00:24	important as it lets us make enough DNA quickly is
00:26	that we can actually run experiments . The whole process
00:30	is a lot in common with how a copy machine
00:32	makes copies of a certain piece of paper . To
00:35	make PcR happen , we need to first mix all
00:38	of the ingredients together in a small plastic tube ,
00:40	which is what we're looking at here on the left
00:43	. So we're going to zoom in on this tube
00:45	and take a look at each ingredient . The first
00:48	thing we need is a buffer . So the buffer
00:50	is just mostly water with some ions in it .
00:52	It's designed to keep the ph constant so the rest
00:55	of the ingredients can do their actual jobs . The
00:58	next thing we need to add into the mix is
01:00	the nucleotides so that we can use them to make
01:03	DNA . The way to think of this is like
01:06	the paper and ink of a copy machine . It's
01:08	the raw material . Remember that we have four DNA
01:11	bases , adenine , thiamine , cytosine and guanine .
01:17	Also be sure to remember the adnan always pairs with
01:20	I mean and cytosine always pairs with wining . This
01:23	is how we're going to use this in a little
01:25	while to actually make our copies of DNA . The
01:29	next thing we want to add into our mix is
01:31	the DNA template . The way to think about this
01:34	is that this is the original copy that you would
01:36	put into a copy machine . Remember that DNA has
01:39	a direction which we call five prime 23 prime .
01:43	And that the strands are anti parallel so that the
01:45	five prime end always matches up of the three prime
01:47	end of the other strand and vice versa . The
01:51	next piece that we needed to add in our the
01:53	pcr primers . These primers are small pieces of DNA
01:57	, which marked the end of the segment of DNA
01:59	. We want to copy , it tells the reaction
02:02	where to start and where to stop . So let's
02:04	say that we want to amplify this entire small piece
02:07	of DNA that we have here in our reaction .
02:09	Then we will need one short primer which will attach
02:12	to each end of the DNA and be oriented in
02:14	the correct direction . The last thing to add into
02:18	our reaction is called the police . To raise the
02:21	primaries is like the copier itself . It is the
02:24	machine that will actually use the DNA bases , the
02:27	template DNA and the primers to make copies of the
02:29	DNA strand . Now we're ready to set up our
02:32	reaction here , we have our reaction in the tube
02:35	and we're now putting it in this machine which is
02:37	called a thermal cycler . All the thermal cycler does
02:40	is change the temperature of the tube after a set
02:42	amount of time . It's this temperature change that's going
02:45	to allow the plumber is to do its work .
02:48	So here we are back in the tube and you
02:50	can see all of our components from before labeled the
02:53	top except for of course the template DNA strand which
02:56	is at the bottom . I've made the template a
02:58	bit longer in this case so we can really see
03:00	each step occurring . Remember we have multiple copies of
03:04	all of these components , not just the one copy
03:06	of each have stuck up the top as a legend
03:09	. You also notice that the temperatures is in the
03:11	upper left corner and right now it's at 18°C, which
03:15	is right around room temperature . This is where things
03:17	start when we turn the thermo cycle or on .
03:20	Now we're going to enter the first step , which
03:22	is called melting . We've now raised the temperature 95°C,
03:27	which is just under the boiling point for water .
03:30	This breaks apart the hydrogen bonds , which hold the
03:32	DNA strand together to give us two separate complementary strands
03:36	of DNA . We now lower the temperature for the
03:41	kneeling phase . In this case I picked 55° but
03:45	it can vary depending on the DNA . You're trying
03:47	to amplify . This is called the kneeling step because
03:51	the primers can now come in and bind or kneel
03:54	to the complementary single strand at the place where they
03:57	match . As shown here , they are now ready
04:00	to be copied . We now enter the final step
04:04	elongation . In this step , we raise the temperature
04:07	to 72°, which allows the preliminaries to recognize the primers
04:11	bound to the single stranded DNA . The polymerase binds
04:15	and begins moving down the existing strand of DNA ,
04:18	always adding the complementary base to the three prime end
04:21	of the growing DNA strand . So as the preliminaries
04:24	comes off as it reaches the end of the strand
04:27	, we now have to complete double stranded DNA molecules
04:30	from what was just one double stranded DNA molecule before
04:35	. So this process can be repeated over and over
04:37	again . Here we've zoomed out with each line now
04:40	representing a single strand of DNA . As we go
04:43	through the multiple cycles , we increase the total number
04:46	of DNA molecules by a factor of two each round
04:49	. So with every round we get more and more
04:51	DNA copies of our target . This is how PCR
04:54	generates copies of a piece of DNA for researchers to
04:57	use in their experiments , and that's the basics of
05:00	how PCR works , Thanks for listening .

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