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Bioquímica

Biologia molecular

DNA

RNA

biologia

aula

videoaula

EaD

Carboidratos

proteinas

lipídeos

genética

engenharia genética

bio

professor

doutor

PCR

polimerase

00:00:03

Hello everyone, welcome everyone,

00:00:05

I'm Professor Thiago Benoliel

00:00:07

and today I brought you an explanation

00:00:10

of one of the most used techniques

00:00:12

in the molecular biology laboratory,

00:00:15

the well-known PCR technique called

00:00:18

polymerase chain reaction, this is the

00:00:22

technique, folks. It was created in the early

00:00:24

80s, in 1983 by a

00:00:28

researcher called Hermes Keri,

00:00:31

he was a researcher in the area of

00:00:35

DNA, biochemistry, but he was

00:00:39

developing a method that was more

00:00:42

practical for isolating genes

00:00:45

what do you mean I have 1 g long huge right

00:00:48

10 thousand genes 20,000 genes but I don't

00:00:51

want them all I want just one so when

00:00:54

you extract dna from an

00:00:56

organism then extract dna from my

00:00:58

cells here I took a skin cell

00:01:00

I extracted dna and then I have to

00:01:03

win and it's all in a

00:01:05

little bit of this size I can't

00:01:07

see it I can't really know what's

00:01:09

there so how can

00:01:12

we isolate a genius is a specific piece of DNA

00:01:15

from the middle of this sea of dna

00:01:18

in the past it was even possible but it was

00:01:21

using a very complicated method so it

00:01:23

took a long time and required a

00:01:25

large investment so it was a lot of work for

00:01:29

you to be able to do this isolation,

00:01:31

sometimes you worked for a month or two just

00:01:33

to be able to isolate the

00:01:35

specific fragment of dna and the hair was

00:01:37

working with this, he ends up

00:01:39

developing this technique that allows

00:01:41

the same work that would take

00:01:44

two, three months to do, you can do

00:01:47

in a few hours, so it is a work that

00:01:52

allowed genetic manipulation and

00:01:55

analysis of rna dna to be much more

00:01:58

efficient than it was the right time,

00:02:01

no wonder

00:02:03

and he ends up selling the patent for this

00:02:05

technique, he becomes a millionaire with it and

00:02:08

then he won the Nobel Prize in 1993,

00:02:13

ten years, right, after the production of the

00:02:16

technique itself, right? He is a Nobel Prize winner in

00:02:19

chemistry precisely because of pcr technique and

00:02:22

then boys, over time the person

00:02:25

ended up growing too much, it is a technique

00:02:28

that has greatly expanded the possibilities

00:02:31

of use and today it is used not

00:02:35

only to isolate

00:02:37

genetic engineering geniuses but it is

00:02:39

used to make diagnostic tests,

00:02:42

such as a test for hiv or

00:02:45

2019 or even dengue fever, you can

00:02:49

do it using the PCR, we can

00:02:53

also use a PC to

00:02:55

map characteristics, so

00:02:58

you can find out if a person

00:03:01

is a carrier of a disease.

00:03:03

the doctor if she has any

00:03:04

specific mutation all of this you can do

00:03:07

using pcr is we use pcr to

00:03:10

do dna sequencing so various

00:03:12

sequencing techniques for you

00:03:15

to identify what the sequence of a dna

00:03:17

is based on pcr we use pcr to

00:03:21

do phylogenetic studies which are

00:03:23

those comparisons, right, between the dna of

00:03:26

different organisms of different

00:03:28

species, we can also do this

00:03:30

using pcr to count a living entity and

00:03:32

another, then in the area of

00:03:35

forensic identification, right, in the case of crimes or

00:03:37

even paternity testing,

00:03:40

the pcr, so understand, right, the range

00:03:44

of universes and uses that it

00:03:47

would appreciate, it reached, not for nothing, one they

00:03:50

won his nobel very close to the

00:03:53

publication he created and 83 more until he

00:03:56

wanted it, it was only published in science in

00:04:01

1985, so

00:04:03

this work is from the republic and the His Nobel Prize

00:04:05

was in 93, a very short period, right,

00:04:08

only eight years after the publication he already

00:04:11

received the Nobel Prize and it's no wonder,

00:04:13

this technique really changed the way

00:04:16

we work with DNA, in any

00:04:21

sphere, from the

00:04:23

academic sphere, scientific analysis, clinics in the

00:04:26

forensic area and in several other areas,

00:04:28

right, so pcr is a technique

00:04:31

with great impact and then, what is

00:04:34

the big deal, what is it used for, do you think,

00:04:37

regent? OK, here's the thing,

00:04:43

it's

00:04:46

specific, so you can take a

00:04:49

region of DNA and copy it over and over and over and over

00:04:54

again, when you

00:04:56

copy just this region many times,

00:04:58

you end up isolating it from the rest, so

00:05:02

it's very easy to see

00:05:03

and with it, you identify it, you

00:05:06

separate it from the rest, so like I said, I don't

00:05:09

know, in our genome there are 20

00:05:12

thousand genes, right, and all the rest of the

00:05:15

huge denarc, so you have to go more than DNA,

00:05:17

but I don't want everything, I want just the gene

00:05:20

related to the growth factor,

00:05:21

so today the growth factor region,

00:05:24

which is just it, so how

00:05:26

can I do this by making a

00:05:29

specific pcr for this region of the

00:05:32

growth factor, can you copy it over

00:05:35

and over and over again millions

00:05:38

of times and then by copying so many times I

00:05:41

end up with several little pieces of this

00:05:43

region several millions ago and now I

00:05:46

have millions of pieces of this region and

00:05:49

the rest of your DNA there in the middle but

00:05:52

wow how do I have so many copies of this

00:05:54

region that I made right, it's one that I

00:05:56

was left with, I can separate it without

00:05:59

any difficulty from the rest, so

00:06:02

using the electron,

00:06:03

the prediction of other techniques is yours and

00:06:05

for that from the rest, it's over, now you have it,

00:06:07

your genius, that fragment

00:06:09

of pure DNA of yours, look, then, really the

00:06:13

pcr makes it possible to do this and imagine

00:06:16

this is used for a

00:06:18

life hiv diagnostic test you will do

00:06:21

a pcr against what a pcr to

00:06:24

amplify the genome of the virus then you

00:06:28

get the specific region that only the virus

00:06:30

has that our cells do not you

00:06:33

collect a sample from the patient's blood

00:06:35

and go there and make a person and with

00:06:37

that point if you have an

00:06:40

amplification if in fact, right, the earth and it

00:06:43

works and you have several copies of

00:06:46

that virus dna it is because in the

00:06:49

patient's blood yes a virus because if there

00:06:51

wasn't there wouldn't be the dna so you know,

00:06:53

you know it wouldn't work if the

00:06:56

patient didn't have the virus inside them

00:06:59

so we use it as a form of

00:07:01

detection if there is

00:07:03

and they are the dna was copied several times it's

00:07:06

because it was in the patient so he

00:07:09

has that disease he is a carrier of that

00:07:11

virus, okay, so that's the

00:07:14

basic logic, right, and that's how it works,

00:07:18

right?

00:07:27

a very

00:07:31

complex, well-elaborated process, right? It's where you

00:07:34

have an enzyme called dna

00:07:37

polymerase, right dna polymerase, and this

00:07:41

enzyme can use a template dna strand, it does

00:07:44

n't have the single strand to make

00:07:48

the copy of the double strand, that's the

00:07:51

action of the enzyme makes dna polymerase, and

00:07:55

then it uses some, you know, some criteria

00:07:57

to do this, but basically this

00:07:59

idea, so a dna polymerase enzyme,

00:08:03

I don't have a simple life, and it makes the

00:08:06

copy of this dna, right, until it gets there in the

00:08:10

region that really encloses it, right?

00:08:13

An important detail is that the enzymes

00:08:14

always copy the DNA of the five-line three

00:08:17

lines, so the new strand that is being

00:08:20

built is what you are generating,

00:08:22

so it is a strand that is always made from the

00:08:25

five lines, for the three lines it is

00:08:27

using another template tape, as

00:08:31

an example, right, for her to do it

00:08:35

herself, okay, so we invented this

00:08:38

process that doesn't even remind us to

00:08:40

study how replication

00:08:42

inside the cell is, well, we imitated

00:08:45

this process, but we did it in

00:08:47

glass so the pcr technique is an

00:08:49

in vitro technique you remove the dna from the

00:08:52

cells you work everything in a

00:08:54

test tube there are no cells

00:08:56

personally involved in the test tube and so for

00:08:58

that we needed some of these

00:09:01

things from the cell, right, it

00:09:03

's the first The thing we need

00:09:06

to make a PCR is for you to have the enzyme

00:09:10

which is DNA polymerase II, what is

00:09:17

certain is that we have to learn it, it is

00:09:22

capable of making the copy of DNA, but there is

00:09:24

one detail, right,

00:09:26

and you are going to do it in vitro, we are done.

00:09:30

manipulating this system so that it

00:09:33

works with temperature variations,

00:09:36

right, in the next class I will explain the

00:09:39

mechanism a little better and you will understand

00:09:41

this issue of temperature variation better,

00:09:42

but see, for it to work,

00:09:45

we have to heat up the dna to cool down, to

00:09:47

heat up, it stays, right, in a

00:09:49

heat-cool cycle, but that's the problem,

00:09:51

enzymes normally can't handle that

00:09:53

when you heat it up, it says natural and

00:09:55

[ __ ] on it, it doesn't work anymore, so then

00:09:58

a new thing came along, right, and it was right

00:10:01

after the publication of myles in 86,

00:10:05

people, he Could you think of a way to

00:10:07

avoid denaturation of the enzyme, what would that be like? It

00:10:09

has to be an

00:10:12

enzyme that can withstand heat, the first

00:10:15

enzyme that was isolated and that could withstand it is over, right?

00:10:23

it was

00:10:25

called

00:10:26

and then the side there in the park el

00:10:30

listón in those gamers, right, what is there,

00:10:33

then this thermus aquaticus enzyme

00:10:36

became known as tati, so that's where it

00:10:40

comes from, right, probably anyone who has

00:10:42

worked with pcr has heard of it, right, it's the

00:10:45

idea of attack attack is a dna

00:10:47

polymerase that was isolated in these

00:10:49

individuals thermus aquaticus and what is

00:10:51

special about it it can withstand heat

00:10:53

you can boil water at 100 degrees and it

00:10:56

remains full it will not

00:10:58

denature it will not stop

00:10:59

working correctly most of the time of the arts and

00:11:02

at the ideal operating temperature in the

00:11:04

range of 72 degrees to 72, but it can

00:11:10

vary depending on the origin of the enzyme and

00:11:13

nowadays you have different types of

00:11:15

dna polymerases that are resistant to

00:11:18

heat, so we don't necessarily

00:11:20

use nowadays the tati it was the

00:11:23

first used it is still used today

00:11:25

it is cheap

00:11:26

asus produces but today there are

00:11:30

other options on the market ok so

00:11:32

this one but a little different more than

00:11:34

logic even dna polymerase

00:11:36

heat resistant very well that is more

00:11:39

We need one, ok, first of

00:11:43

all, we build a

00:11:46

new one, right? So, she has a strand of DNA

00:11:49

template and she builds a new DNA

00:11:53

from this template, so if there's a

00:11:55

cytosine, she puts it in a girl, if there's a

00:11:58

little girl, she puts it in an adenine is like that, right?

00:12:18

the four nucleotides

00:12:21

right adenine cytosine guanine and thymine

00:12:25

you have to have all four so in your

00:12:28

pcr reaction you also need to

00:12:30

include the four nucleotides but it is

00:12:33

a detail for them to work they

00:12:35

have to be in the form of triphosphate which you do

00:12:38

n't remember that after the carried out

00:12:40

in this, the nucleotides can be in

00:12:43

the phosphate or triphosphate form, which

00:12:47

the polymerase used is in the

00:12:50

triphosphate form, so we need

00:12:54

the green ones, we call them in 3d, they are

00:13:00

the deoxyribose nucleotides because

00:13:03

I'm making a DNA, right? triphosphates,

00:13:06

right, there are four, right, people, it's atp,

00:13:11

ttp, gtp, and the last one is

00:13:20

ctp, so the four nitrogenous bases,

00:13:24

right, adenine, thymine, guanine and cytosine,

00:13:28

so, in addition to dna polymerase, we

00:13:30

need to use this, right, these, these

00:13:35

are everyone has to be in the reaction,

00:13:37

what else do we have to put into the

00:13:39

reaction, come on, let's go from top to bottom,

00:13:40

how do we need to put people, the DNA

00:13:46

mold, right?

00:13:55

Is it right, what

00:13:59

do you mean, people, if I want to do an

00:14:02

amplification of the DNA of a bacterium, I don't know,

00:14:04

it's a gene from a bacterium, how is

00:14:07

it going to amplify this piece, that one, is it

00:14:09

going to copy this piece? If I don't have

00:14:10

this DNA, you have to have it, so there is you have to

00:14:13

have at least one copy of it, right, you

00:14:16

have a copy of it using your PC and you

00:14:18

can copy it millions of times and come out

00:14:21

with millions of copies, but you have to

00:14:24

have an original copy,

00:14:26

you have to have one to do it with, right? the

00:14:29

other copies, you know, so if I'm going to

00:14:31

make a PCR for a genius, bro, I

00:14:34

need to win a year, I'm going to make

00:14:36

one,

00:14:37

it's from a mouse gene, I need to beat a

00:14:39

rat gene, and there you go, so whatever

00:14:42

the target is, you need to have a copy

00:14:46

of it, you're pure, no, like I said,

00:14:49

it can be mixed, it can even be

00:14:51

inside the cells, sometimes it depends

00:14:54

on the type of cell, we can do

00:14:56

PCR even without you extracting the

00:14:59

DNA, so sometimes I get just a

00:15:01

little bit of it. bacteria or tissue that

00:15:04

you want to do the PCR and put it in the

00:15:07

reaction, as in the reaction you heat it up

00:15:09

a lot, the cells end up having it and then you

00:15:12

already have free DNA, so often

00:15:15

this one, this one, the mold doesn't release in

00:15:18

pure form, it can be mixed.

00:15:19

other things, there's no problem if there's a problem

00:15:22

there, if there's this win there, a

00:15:25

well-made person is capable of detecting it now, team,

00:15:28

making the copy several times, beauty, so

00:15:30

they're another, because there's another other

00:15:34

ingredient here, we need it, right, and

00:15:36

we have

00:15:37

the sound

00:15:39

and how does dna polymerase

00:15:44

know exactly which

00:15:48

specific region it is going to copy because dna

00:15:51

has thousands of them, right?

00:15:59

you know that that is the only

00:16:01

small region that it will copy

00:16:03

several times, so to define

00:16:07

the region that dna polymerase will copy,

00:16:10

we use small single-stranded dna

00:16:15

that we call oligonucleotides,

00:16:18

right, and these bind it, oligonucleotides,

00:16:22

they will recognize the ends of the

00:16:26

region you want to copy, so for

00:16:29

you to copy a region of DNA you will

00:16:31

need at least a pair of these

00:16:34

oligonucleotides, one that will anneal in

00:16:37

a region at one end, the other that will

00:16:39

anneal at the other end and then between the two

00:16:43

dna polymerase will make this copy until

00:16:46

the mechanism is how it recognizes

00:16:49

why they delimit, we will

00:16:51

see in the next class, the next video

00:16:53

will be precisely showing this, but

00:16:55

understand that what makes the pcr

00:16:59

is specific is precisely this

00:17:02

legal pair of nucleotides that we put

00:17:05

there in this region, you know, as we just

00:17:08

called clothes, which means they are

00:17:10

small in the range of 18 20 nucleotides,

00:17:14

it could be a little more, a little less,

00:17:16

but it is in this range, so very small,

00:17:17

it's ready to gain 20 other types is not enough

00:17:21

and it is generally single stranded it does not

00:17:23

need to be double stranded, you

00:17:25

just take a small piece made in single strands and it will

00:17:27

be complementary to the region you want to

00:17:30

amplify, so if I want to copy the

00:17:32

DNA from here to here I need a

00:17:34

tongue one that will connect to this end,

00:17:37

another that will connect to this end, right, or

00:17:39

I love using the name in English, which is

00:17:42

primers, so strimers, they are

00:17:45

exactly these oligonucleotides that

00:17:48

delimit the tip of the region that will be

00:17:51

copied, calmly, so they have to

00:17:53

be here in the reaction. Also, let's go

00:17:56

this way, Igor,

00:18:02

and in the English beach, someone calls it a

00:18:15

beach, it's like a fire, a pot allows

00:18:18

you to have a conventional PCR, a pair of

00:18:21

nucleotides are necessary, one that the

00:18:23

line doesn't point to, the other that the line at the

00:18:25

other end, okay? As I said, as they

00:18:28

are small, 20 nucleotides, this is

00:18:31

very easy to synthesize, listening to

00:18:33

chemistry, so by carrying out chemical reactions

00:18:37

in sequence, we can

00:18:39

even artificially synthesize these

00:18:42

tiny nucleotides, it is very easy to

00:18:43

do and there are companies that

00:18:45

specialize in this, so you just order them.

00:18:48

the sequence says, look, I want a

00:18:50

prime minister, pain or not, it's an

00:18:52

oligonucleotide, and it's

00:18:53

with this sequence here, PC GTA

00:18:56

sends you to the company, they send you

00:19:00

back in a week, okay, so quickly,

00:19:01

you receive these oligonucleotides that

00:19:04

you need, calmly So this

00:19:07

idea is for you to make a PCR, of course the

00:19:10

PCR conditions have to be ideal,

00:19:12

so the DNA polymerase has a buffer

00:19:15

and comes with it, so when you

00:19:17

buy it, the buffer already comes so you

00:19:20

can use it together, right? the buffer

00:19:23

also has to be placed in the reaction and

00:19:25

you will need the machine so

00:19:28

type a machine that regulates the

00:19:31

temperature change cycles as I

00:19:34

said this in vitro process is

00:19:36

controlled by changes in temperature

00:19:38

you heat the dna cools it heats and

00:19:41

cools and then you have the

00:19:44

PCR process happening and then you're not going to do

00:19:46

it by hand, right? You're not going to go back there with three

00:19:48

pans of water at temperature and

00:19:49

cutting tubes, right, bro, no,

00:19:52

seriously, it could even be done in it's

00:19:53

already started today, we have one equipment

00:19:57

a machine called a thermocycler and the

00:20:00

thermocycler will carry out this reaction

00:20:03

for you, then you prepare the reaction,

00:20:06

mix it all here inside the tube,

00:20:08

boss, plug, put the water in, adjust the volume,

00:20:10

right, put it in the tube, this tube goes

00:20:13

inside this machine, close the machine,

00:20:16

program the cycle just look I want it to

00:20:18

make so many circles and such a temperature like

00:20:20

this here and the whole machine

00:20:23

programs it there press the button let it be

00:20:26

there on the machine it will make this oscillation the

00:20:28

temperature comes out the way you

00:20:30

programmed it is called a thermocycler zé

00:20:34

and so on On average, a PCR reaction lasts

00:20:38

around one to two hours, depending on the

00:20:41

reaction, up to four hours, so the

00:20:44

result is very quick and you

00:20:46

can actually analyze it, you

00:20:48

can already see it, so yes, it

00:20:50

has changed a lot, right, it has improved a lot over those

00:20:53

two three months it took for

00:20:55

us to be able to isolate a region of

00:20:57

a dna, so this morning you

00:21:00

can already have a consistent result,

00:21:02

so that's it, so this

00:21:05

general idea about pcr, in the

00:21:08

next video, I'll discuss a

00:21:10

little about the mechanism and then you

00:21:12

will understand a little better how

00:21:13

this pcr reaction really works.

Description:

Quinta aula do Módulo Básico sobre Engenharia Genética.

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Which format of "Engenharia Genética - Aula 05 - PCR" video should I choose?

The best quality formats are FullHD (1080p), 2K (1440p), 4K (2160p) and 8K (4320p). The higher the resolution of your screen, the higher the video quality should be. However, there are other factors to consider: download speed, amount of free space, and device performance during playback.

Why does my computer freeze when loading a "Engenharia Genética - Aula 05 - PCR" video?

The browser/computer should not freeze completely! If this happens, please report it with a link to the video. Sometimes videos cannot be downloaded directly in a suitable format, so we have added the ability to convert the file to the desired format. In some cases, this process may actively use computer resources.

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How can I download an audio track (music) to MP3 "Engenharia Genética - Aula 05 - PCR"?

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How can I save a frame from a video "Engenharia Genética - Aula 05 - PCR"?

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What's the price of all this stuff?

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