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Lecture Comments (6)

0 answers

Post by Mena Jirjees on January 4, 2015

Why after the first two cycles of PCR are there zero target DNA ?

1 answer

Last reply by: Dr Carleen Eaton
Wed Mar 26, 2014 6:35 PM

Post by Sarah Ferreira on February 23, 2014

Lecture will not play for PCR section. The lecture will just stop and start over all the way from the beginning.

0 answers

Post by Naomi Harris on November 29, 2013

Hello,
If bacterial DNA is mentholated, how can a restriction enzyme cleave it?

0 answers

Post by Jonathn Ochoa on October 31, 2013

Any DNA Fingerprinting examples? We are doing a lab in school and I need a little help.

0 answers

Post by Billy Jay on April 12, 2011

I understand that you if you combine Plasmids with DNA (containing the gene of interest), and treat them both with the appropriate restriction enzyme (and DNA ligase), a recombinant plasmid would be produced. However, my question though is how do you remove and isolate the plasmid from a bacteria cell to begin with?

Biotechnology

    Coming soon

Biotechnology

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Definition of Biotechnology 0:08
    • Biotechnology
    • Genetic Engineering
    • Example: Golden Corn
  • Recombinant DNA 2:41
    • Recombinant DNA
    • Transformation
    • Transduction
    • Restriction Enzymes, Restriction Sites, & DNA Ligase
  • Gene Cloning 13:48
    • Plasmids
    • Gene Cloning: Step 1
    • Gene Cloning: Step 2
    • Gene Cloning: Step 3
    • Gene Cloning: Step 4
  • Gel Electrophoresis 27:25
    • What is Gel Electrophoresis?
    • Gel Electrophoresis: Step 1
    • Gel Electrophoresis: Step 2
    • Gel Electrophoresis: Step 3 & 4
    • Gel Electrophoresis: Step 5
    • Southern Blotting
  • Polymerase Chain Reaction (PCR) 32:11
    • Polymerase Chain Reaction (PCR)
    • Denaturing Phase
    • Annealing Phase
    • Elongation/ Extension Phase
  • DNA Sequencing and the Human Genome Project 39:19
    • DNA Sequencing and the Human Genome Project
  • Example 1: Gene Cloning 40:40
  • Example 2: Recombinant DNA 43:04
  • Example 3: Match Terms With Descriptions 45:43
  • Example 4: Polymerase Chain Reaction 47:36

Transcription: Biotechnology

Welcome to Educator.com.0001

We are going to finish out the series of lectures on molecular genetics with the discussion of biotechnology.0003

We will start with the definition of biotechnology. Humans have used biotechnology techniques for thousands of years0009

for example, selective breeding to get a plant with particular qualities like a tree that makes larger apples or using yeast to make bread rice.0018

Those are examples of biotechnology.0030

Looking at the formal definition, biotechnology is the use of organisms, components of organisms0033

or biological processes to make industrial products or to perform industrial processes.0041

Though in the broadest definition, biotechnology does include examples such as selective breeding or the use of yeast like I talked about.0048

Now, though, a lot of times when the term biotechnology is used, people are referring to more molecular biology techniques in particular genetic engineering.0057

Genetic engineering is a branch of biotechnology that involves the manipulation of genes,0072

which will result in a change in the genetic composition of an organism.0078

Genetic engineering involves the manipulation of genes and/or changing the genetic composition of an organism.0083

Through genetic engineering, we can get bacteria to do work for us and produce products.0105

And genetically altered bacteria have been used in the clean-up of oil spills for example.0113

There is a type of corn that was created. It is called "golden corn".0118

This plant produces corn that contains beta-carotene. Beta-carotene is a precursor of vitamin A.0126

And there are numerous examples: insulin, biotechnology is used to produce insulin; to produce immunizations such as hepatitis B immunization.0139

Today, we are going to focus on some of the commonly used techniques in laboratories for biotechnology.0153

And we are going to start out by just revisiting the topic of recombinant DNA.0161

Recall that when DNA is recombinant, it is comprised of DNA from two different sources.0166

A lot of times, what will be done in the laboratory is we will express a gene from an organism in a bacterial cell.0178

We might want to make a particular protein that is produced in humans, and we will put that gene into that bacteria.0188

And then, the bacteria will produce that protein as well as its own bacterial proteins.0196

Transformation, recall, is a process through which bacteria can take up DNA from an outside source.0207

We talked about that in an earlier lecture. This is something that occurs in nature and in the labs.0226

Laboratory will use processes such as the heat shock method to make bacteria competent.0230

In the process of transformation, bacteria take up DNA from the environment. Bacteria that we call competent are able to take up DNA.0236

And one AP Biology that you may have done or will do involves the heat shock method to make bacteria competent to perform transformation.0254

Another way to get foreign DNA into a bacterium is through transduction.0265

In this case, we use phage to carry DNA into bacterial cells, and this occurs in nature as well because sometimes a virus, a phage will infect a bacterial cell.0270

And it will accidentally package some bacterial DNA in its capsid, carry that DNA to another bacterial cell and then, inject it.0287

Now, let's focus on ways in which we can...once we get the foreign DNA in,0296

how can we make a combination of bacterial DNA and human DNA or whatever type of DNA we are trying to express?0305

We need a way to actually put the DNA in.0315

Well, before we get to that, you need to understand restriction enzymes because this is a way of cutting DNA.0322

And if we can cut the DNA, we could paste in the different segments of DNA.0328

Restriction enzymes are actually found in bacterium naturally, and they have been isolated from bacteria.0333

Restriction enzymes cleave DNA at particular sites called restriction sites.0342

Restriction sites are usually about 4 to 8 nucleotides long, and they are specific sequences.0353

Within the site, a certain restriction enzyme, it will target a certain site, a certain sequence, and it will only cleave at a particular place within that site.0363

Now, if you think about this, bacteria have these enzymes within them.0374

For example, there is one that is used frequently in the lab. It is called EcoRI.0381

And it is called that because it is named after E. coli, which is where it was isolated from.0386

If an E. coli cell has this EcoRI floating around in it, you would think that it would go around and chop up the bacterial DNA, which would not be useful.0391

The purpose, though, of the restriction enzyme is not to chop up the bacterial DNA. It is to chop up the DNA of an invader such as a phage.0402

So, if the bacteria is infected by a phage, and there is phage DNA within the cell, EcoRI could cleave that DNA.0411

However, if you think about it, the bacteria need to protect its own DNA, and the way it does that, one way is to methylate its own DNA.0419

A bacterial cell will have methylated DNA so that causes the restriction enzyme not to be able to attack its own DNA,0427

yet, to still cut up the DNA of other organisms such as phage, so that is the background on where these come from.0435

Now, let's focus in on EcoRI as an example because it is a very frequently used restriction enzyme.0445

EcoRI has a target site or restriction site with this sequence that recognizes the C-T-T-A-A-G, and it cuts the DNA between this A and G.0451

That is where the cut is made. Let's look at an example.0471

If we had a double-stranded DNA, and we had the DNA going along, and then, in some areas of the DNA,0477

there would happen to be this sequence, we are talking about target DNA.0484

The complimentary strand for the double-stranded DNA would be C-T-T-A-A-G.0491

If you put EcoRI in a mix with this DNA, and you let it do its work, it is going to cut the DNA here because we still have this sequence.0502

You see, it starts over here, C-T-T-A-A-G, but it still this sequence. EcoRI recognized it.0514

Here, again, C-T-T-A-A-G, it is going to cleave between the A and the G.0520

Now, let's see what we are going to end up with. We are going to end up with the 5'-end with the G.0529

And then, we are going to end up with A-A-T-T-C and the 3'-end.0535

3'-end, we are going to end up with C-T-T-A-A, and then, over here, the G and then, the 5'-end.0542

Ends like this with these overhangs are known as sticky ends.0554

There are restriction enzymes that because of the particular sequence they target,0560

cut in such a way that you end up with blunt ends that they just cut across, and the ends are blunt.0566

Particularly useful, though, are the restriction enzymes that cut DNA and leave sticky ends.0574

The reason this is important is that these fragments of DNA can ligate or attach to another piece of DNA with complimentary sticky ends.0582

Actually, let's say this is bacterial DNA. This in black, this is bacterial DNA.0598

And I am trying to get DNA from another organism into the bacterial cell so I can study it.0604

What I can do is take some foreign DNA, foreign to the bacteria, and I could also cleave it with EcoRI, so 5' G-A-A-T-T-C 3', 3' C-T-T-A-A-G- 5'.0613

EcoRI is added. It is going to come along and again, is going to cleave right here and right here.0643

Now, what I have got is - let's write this over here the way it is going to end up with its sticky ends - G-A-A.0651

Actually, the cleavage was right there, so we are just going to have G on this side, and we are going to have A-A-T-T-C 3'.0663

Down here, we are going to have 3' C-T-T-A-A and over here, the G and the 5'-end.0674

Now, it is possible that if I mix this DNA with this DNA, one thing that could happen is these two could just religate. These two could go back together.0681

It is also possible that this could ligate to this, and you see how it could fit together like a puzzle.0692

And what I would end up with, then, is 5', the bacterial DNA, G.0701

So, if I am taking this 3'-end, I have this fragment here, C-T-T-A-A, ligating with this fragment, A-A-T-T-C 3'-end.0711

OK, then, what I am going to end up over here is G and then the rest of the fragment.0735

You see what I ended up with. Well, what I started with was bacterial DNA that I cut with the restriction enzyme.0749

Foreign DNA, DNA from another organism that I cut with the same restriction enzyme, I ended up with these two fragments of DNA.0755

This one is complimentary to this one, and they were able to ligate together.0768

This also, in order to get them to actually seal together, requires an enzyme called DNA ligase.0775

I would have to mix my DNA with this enzyme, DNA ligase,0781

to end up having the two, not just temporarily hydrogen bond, but to actually stay together, so DNA ligase.0785

This fragment could, then, do the same thing with this fragment. The result is recombinant DNA.0795

So, this piece of DNA down here is recombinant. It consists of DNA from two different organisms.0805

Restriction enzymes are an extremely important tool in cloning, so let's talk now about gene cloning.0823

Gene cloning or DNA cloning is the production of multiple identical copies of DNA.0832

If we want to study a protein, or we want to produce a particular protein or other product from a cell, and we want to produce it in bacteria,0838

we are going to want to produce multiple copies of that gene, and we are going to use gene cloning or DNA cloning.0851

And this is where the restriction enzymes come into play.0857

Let's go ahead and go through the steps of gene cloning.0861

One commonly used method of cloning involves the use of plasmids.0864

Recall that the bacterial chromosome is a single circular piece of DNA.0869

However, bacterial cells can have small circular elements of DNA outside that main chromosome that carry a few genes.0876

For example, they may carry a gene for antibiotic resistance.0884

We can use plasmids as a vector. Now, what is a vector?0889

A vector, when we are talking biotechnology, it is a segment of DNA that is used to carry donor DNA.0896

Here, I have the bacterial cell. This is the host cell.0913

This is a bacterial cell, and this particular bacterial cell or in reality, what I would have is many, many bacterial cells.0916

So I would take a whole lot of bacteria, and I would take a type of bacteria that did not have plasmid for simplification.0924

And then, here, I have the cell containing...here is a eukaryotic cell, and it has got its own DNA.0935

Here is the nucleus. Here is the donor DNA.0944

And if I want to study certain genes in here, I want to use this bacterial system to make many copies of the gene I am interested in.0952

The first step is going to be to get a plasmid. Get many plasmids, and that is what these are right here.0967

We will just put it like this, all my donor DNA, and I am going to cut both of these with a restriction enzyme, cut with the same type of restriction enzyme.0978

Just to note, you might hear restriction enzymes called restriction endonucleases. That is just a different name for the same thing.0997

If I cut these, what I am going to end up, then, with is an opening, so I am just going to cut that, cut that, cut that.1010

Wherever this particular sequence is, the restriction site is, which is just going to occur many times throughout the genome,1025

I am going to end up with a bunch of pieces of DNA.1031

Because I cut with the same restriction enzyme, these ends will be complimentary to the ends that I have left on this plasmid.1037

So, I cut with the same restriction enzyme just like I should do with EcoRI.1046

I am going to end up with sticky ends here, complimentary sticky ends here.1050

First, cut DNA, so host DNA actually, a donor DNA and plasmids with the same restriction enzyme.1056

The next step is going to be to combine the restriction fragments that I have from cutting up this donor DNA,1078

combine restriction fragments, plasmid DNA and DNA ligase.1081

What is going to happen next is some of these plasmids will just reseal. That is definitely possible.1102

So, I might get some plasmids with the DNA ligase that just reseal.1110

However, what will happen with many of the plasmids is that they will end up with a fragment of the donor DNA within them.1113

These are recombinant plasmids.1126

Next, I need to get these plasmids into the bacterial cell, and I will do that through transformation; and we talked about different methods.1133

We are going to transform the plasmids into the bacterial cells.1143

We talked about methods of transformation for example the heat shock method.1153

I am going to combine the bacteria with these plasmids, some of which are recombinant.1158

I am going to go through the procedure, transform the bacteria, and then, what I will end up with is this.1163

I will end up with some bacteria that have plasmids that are not recombinant,1172

some bacteria that have recombinant plasmids and some bacteria that have no plasmid.1178

The next step is to figure out which bacteria are carrying the plasmids with the recombinant DNA and which DNA in particular I am interested in.1187

And there is techniques to do this, and a lot of it has to do with constructing the plasmid.1201

Going back to the beginning, what you want to do is use...one way to approach this would be to use a plasmid with genes for antibiotic resistance.1206

Let's say this plasmid also has a gene for resistance to an antibiotic such as ampicillin.1223

So, I will call this antibiotic resistance gene, so these plasmids are carrying that.1231

If I take the bacteria after transformation and plated it out and grow it on agar, I will get colonies.1242

In this medium I used with the nutrients, I am going to include an antibiotic.1253

If you think about what is going to happen, if a cell is carrying a plasmid,1264

and we said all the plasmids we start out with are going to have this antibiotic resistance gene,1271

any cell carrying a plasmid for antibiotic resistance gene should be able to grow on this antibiotic infused nutrient.1276

Cells without the plasmid cannot grow, so I am going to put plate on medium with antibiotic.1289

If I used a plasmid containing antibiotic resistance genes, and then, in the end,1303

I plated the bacteria onto a plate containing an antibiotic, and I see a colony there, I know at least it has a plasmid.1308

I do not know that it has recombinant DNA in it, donor DNA, but at least there is a plasmid there, so I have cut out all these bacteria.1316

The way to figure out which bacteria actually have recombinant DNA is a little bit trickier.1327

And one method that is used is something called a blue white screen.1333

And in this blue white screen, white colonies contain bacteria with recombinant plasmids, so this is going to be white.1339

These colonies will be blue. They contain recombinant DNA.1353

Just briefly, what this involves is using the lacZ gene.1360

We talked about operons. We talked about the lac, and one of the genes on that operon is called lacZ.1366

lacZ codes for an enzyme called beta-galactosidase.1374

And the job of beta-galactosidase in the bacterial cell is to break down lactose, so the bacteria can use that as a nutrient.1380

However, it so happens that there is a molecule similar in structure to lactose called X-gal.1387

When X-gal is hydrolyzed, it produces a blue product.1395

Beta-galactosidase can use X-gal as a substrate, and therefore, catalyze this reaction where X-gal produces a blue product when it is hydrolyzed.1400

It is going to cleave the X-gal and produce blue.1413

If we started out with a plasmid, so we said it has antibiotic resistance gene on it, well, we can also use a plasmid that contains the lacZ gene.1419

And we are going to put that lacZ gene in such an area that the restriction site will be similar in there.1439

So, when I cut this plasmid with a restriction enzyme, I am going to disrupt the lacZ gene.1447

If the plasmid just religates to itself, it will go back to being intact. It will have functional lacZ.1455

However, let's say recombinant DNA ends up in here, then, I am going to have my lacZ gene,1463

some of the foreign DNA in here, that lacZ gene is not going to function anymore.1471

In the original plasmid, there is a functional lacZ gene.1481

If recombinant DNA ends up inserted within that sequence, the lacZ gene is disrupted.1484

If I plate these bacteria on a plate that contains antibiotics, I just get colonies containing plasmids.1494

If I also include X-gal on here, those colonies containing bacteria with intact plasmids, not recombinant, will end up blue.1502

The lacZ gene is working fine. It can hydrolyze X-gal and make that blue product.1514

So, I will all these blue colonies, and these are the ones that do not contain a DNA I am interested in.1524

If you look at this cell, this cell has recombinant DNA in it. It is disrupting the lacZ gene.1538

Since it will not be making beta-galactosidase, it can hydrolyze X-gal, and those colonies will be white.1543

So, I look down on the colonies, I say "OK, the ones that are white are the ones that I want.".1549

They are containing the foreign DNA that I wanted to introduce to those cells.1555

When I look at all these colonies together, what I have essentially created is a DNA library.1561

When I take all these plates with all these mini-white colonies, I say "Alright, each of these white colonies contains a particular piece of1568

donor DNA that has been replicated, and now, I have got a whole library of DNA that I can study.".1578

Finding the DNA that I am interested in, it can be done using what is called a probe.1588

A nucleic acid probe will contain a sequence that is complimentary to the DNA I want to study.1595

So, if I am studying a certain gene, and I even just know part of that sequence or something, I could make a probe, DNA or RNA,1607

and I could radioactively label it, or label it in such a way that it fluoresces or color change or something,1614

so that I can, then, screen my library and find the DNA that I am interested in.1622

And there are many techniques for this that are beyond the scope of this course.1626

But for right now, you should be familiar with restriction enzymes, the basics of gene cloning.1630

And just be aware the nucleic acid probes can be used to find a target DNA sequence.1638

Another frequently used procedure in biotech labs is gel electrophoresis.1646

Gel electrophoresis allows DNA fragments to be separated according to size.1654

This is showing you an example of a gel after the procedure has been performed.1675

But if we just start from the beginning and talk about what does this gel consist of, well, it consists of something called agarose, which is a polymer.1680

Up here are wells that the DNA can be placed in.1689

So, the first thing we are going to do is we are going to cut up the DNA we are studying using restriction enzymes.1694

We are going to end up with restriction fragments, and we are going to place some of the DNA in each of these wells.1705

The gel is, then, submerged in a liquid in a machine that can apply an electric current to the gel.1720

At the end near the wells is going to be the cathode, so negatively charged.1740

The anode is going to be at the opposite end, and that is positively charged.1748

When the current is applied to the gel, the DNA starts out in the wells, and it is all these little fragments of different sizes,1755

current is applied, the DNA is going to move towards the positive charge.1764

Recall that DNA is negatively charged. It has negatively charged phosphate groups on it, so it is going to move towards the anode.1768

When we turn that machine on, we leave it for a couple hours to many hours.1777

It just depends on the fragment you are looking for and how large the gel is in various things.1782

You turn that on. Let it run, and then, turn the machine off.1788

Turn the current off.1795

Now, when you just take this gel out and look at it, you are not going to be able to know where the DNA is. You are not going to see all these bands.1797

What you have to do is, after you turn off the machine, add a dye to the gel, and the dye will bind to the DNA. That is what you see here.1804

This dye will actually fluoresce under UV light.1822

So you will take your gel, and you will look at it under ultraviolet light, and that is when you will see the series of bands.1830

And the bands that are farther down that move faster are generally smaller fragments. Larger fragments are up here.1836

And you see a wider band. There is more DNA.1844

There is inner band. There is less DNA there.1847

DNA can actually be recovered from these gels. You can actually cut out a little piece of the gel, put it in a test tube, dissolve the gel and recover the DNA.1850

So, if I know the size of the DNA I am looking for, then, I could say "Oh, OK, this is where it is, and I can actually cut this out and recover it.".1860

Proteins can actually also be used. It can be separated out using gels.1871

Finally, if I am trying to find a certain fragment of DNA, and I do not know exactly where it is here, there is a technique called Southern blotting.1877

And we already talked about radioactive probes and nucleic acid probes.1890

And in Southern blotting, it is a method of using a radioactive probe to detect the DNA of interest, to find out which band contains the DNA of interest.1895

What the electrophoresis does is it separates out DNA or proteins on the basis of size.1916

Then, techniques can be used to detect which band contains the DNA that you want to study.1922

Another technique that you may have heard of is called polymerase chain reaction or PCR. This is a method of copying or amplifying DNA.1932

When we say we amplify it, what we mean is we are copying it, and it uses a machine called a thermocycler. This is the PCR machine.1943

The reason it is called a thermocycler is it takes the DNA through a series of heating and cooling cycles.1955

And during this process of heating and cooling, the DNA of interest, the target DNA, is replicated.1960

PCR is particularly valuable in situations where you have only small amount of DNA.1968

For example, if you are a forensic scientist, and there has been a crime scene; and we recovered a small amount of DNA from the suspect, DNA at the scene,1974

we could amplify that and then, use that to try to figure out who committed the crime.1985

Another example would be if a scientist is studying a small amount of DNA from an animal or a human that lived thousands of years ago.1992

PCR can be used in that situation, as well. In order to perform PCR, you are going to need a few things, though.2001

You are obviously going to need your target DNA. You need the sample of DNA you are trying to amplify.2011

You will also need primers, so if I have a piece of DNA that I got from forensics, and here is my DNA, 5' to 3', 3' to 5', I am going to need a set of primers.2015

One of the primers should be complementary to one end of one strand.2033

The other primer should be complementary to the other end of the other strand - actually down here - because remember that replication will occur 5' to 3'.2039

OK, in red, this is the primers.2049

If I am going to copy or amplify DNA, I am also going to need nucleotides to synthesize more DNA.2056

So, we need primers complementary to sequences on the target DNA actually sequences flanking the target DNA.2062

I am going to need nucleotides, and I am also going to need, in order to synthesize new DNA, DNA polymerase.2070

And you cannot just use any old DNA polymerase because we are taking this DNA through a series of heating and cooling cycles.2076

And a lot of DNA polymerase, most DNA polymerase will not function at high temperatures.2083

However, DNA isolated from extremophiles, which are bacteria that live in extreme conditions, in this case, the extreme condition is heat.2088

They are sometimes called thermophiles.2099

Extremophile bacteria, they live in extreme conditions, and there has been a type of polymerase isolated from these called TAC polymerase.2103

This is a type of DNA polymerase that can function at high temperatures. It is heat-resistant.2116

So, you start your PCR. You have your thermocycler.2125

You have your target DNA. You have got your primers, TAC polymerase and nucleotides.2127

You start things up. The machine will go through a cycling process.2136

The first cycle is the denaturing cycle, so the DNA is double-stranded.2142

We are going to need to denature it and separate that out, so that we end up with two single-stranded pieces of DNA. That is step one.2153

Step two is the annealing phase. This involves heating the DNA up.2169

Next, the machine will cool the DNA down for the annealing phase- cooler.2178

So, the DNA are separated in the denaturing phase. They are separated out.2192

They are no longer double-stranded. It is now single-stranded.2196

Annealing means if something is going to anneal, it is going to stick. The primers, at this point, anneal to the target DNA.2199

I have got a primer that will anneal here and a primer for the opposite strand, and these primers are very specific for my target DNA.2212

That is step two. It is this annealing phase.2223

Step three is sometimes called the elongation phase or extension phase or extension.2227

At this point, TAC polymerase extends or elongates the primer using the target DNA as a template.2238

During the extension phase, I have added nucleotides, and now, I have got nucleotides in there.2258

And TAC polymerase is going to go ahead and make that complementary strand.2269

And that is going to happen with both of these, so I am going to end up with these two copies.2274

Then, the cycle will start again. These two will be denatured, so now, I am going to end up with 1, 2, 3, 4 single strands of DNA.2287

Primers will anneal, and then, TAC polymerase, it is going to extend the primer. The cycle will occur again, and that is how amplification occurs.2299

I started out with one strand of DNA. From that, I ended up with one molecule of DNA.2312

And then, I put two molecules of DNA right here. Then, I am going to end up with denaturing those, synthesizing a new complementary strand.2320

Now, I have got four strands and eight and so on, and that is how the DNA is amplified.2330

And this is obviously a very useful technique, but it does have its limitations.2337

For one thing, only a relatively shorter piece of DNA can be replicated this way.2340

Another issue is that errors can be introduced during the synthesis process.2346

So, this does not replace cloning, but it is another technique we have in our arsenal that has been extremely useful.2353

Finally, we are going to talk about DNA sequencing and in particular, the human genome project.2362

When scientist study in gene, one thing that they often want to do is actually figure out the nucleotide sequencing of the gene.2367

And when we say we are going to sequence the gene, sequencing means we are going to determine the nucleotide sequence of DNA.2373

This is, now, automated. It is done by sequencing machines, and in fact, you may have heard of the human genome project.2389

The human genome project began in 1990, and it concluded in 2003.2397

And this involved many, many labs and institutions working together and sequencing the over 3 billion base pairs of the human genome.2412

And that data was stored in a database so that scientists can use that for further studies.2427

Alright, focusing on some examples regarding biotechnology, example one: describe the steps involved in cloning a gene using a bacterial plasmid as a vector.2442

Remember that if we want to clone a gene, we are trying to make copies of it.2455

And what we are going to need is bacteria, plasmids and various enzymes for the process.2462

And we are also going to need our DNA that we are studying or donor DNA.2470

The first step is going to be to use restriction enzymes to cut the donor DNA and plasmids.2474

So, we are going to use the same restriction enzymes on both so that they have complementary ends.2491

Then, we are going to mix the DNA fragments plus the plasmid that has been cut, and we are going to add DNA ligase to this, so plus ligase.2499

That will allow the DNA to anneal, and that will create some plasmids that are recombinant and contain donor DNA.2519

Next, we are going to transform the bacteria, so we will have to make the bacteria competent and bacteria.2527

So, when we say we are going to transform the bacteria, we mean that the bacteria will take up the plasmids.2541

Finally, we are going to plate the bacteria on a growth medium.2549

After that, we need to determine which colonies contain the DNA that we are interested in, OK?2561

The essential steps: cut donor DNA and the plasmid; mix the DNA fragments, the plasmid and DNA ligase;2570

transform the bacteria, so they are containing the plasmid DNA; and then, plate the bacteria on a growth medium.2577

Continuing on with this in example two: what features could the vector plasmid in the previous example2586

have in order to make it easier to locate those colonies that contain - let's just say that contain - recombinant DNA or with recombinant DNA?2592

How do I know which colonies have the recombinant DNA?2606

Well, the first thing is I want colonies that contain a plasmid. How do I know which colonies even have the plasmid?2609

What I can do, what features use a vector plasmid with antibiotic resistance genes.2620

And then, when I plate the bacteria, I am going to have a growth medium that contains an antibiotic.2633

I know which colonies contain the plasmid. It will be any colonies that grow on this.2643

So my plasmid is going to have antibiotic resistance genes right here, number one.2650

Now, I know these are all plasmids, but which ones contain recombinant DNA?2660

Another feature I want my plasmid to have is lacZ gene on the plasmid in the region where the foreign DNA will insert.2664

If the lacZ gene is here - this is number two, this is the lacZ gene - and the restriction enzyme cuts somewhere in here,2686

and then, the recombinant DNA inserts there, the lacZ gene has been disrupted.2695

And if I also have X-gal on here, only the non-recombinant DNA can hydrolyze the X-gal and turn blue.2708

So, white colonies will contain recombinant DNA, and that will only occur if I put X-gal on the growth medium.2716

Two features I want to have is a vector with antibiotic resistance genes and a vector containing the lacZ gene.2736

Example three: match the following terms with their descriptions- PCR, gel electrophoresis, restriction enzymes and genetic engineering.2744

Starting with PCR: cleaves the DNA at specific sites in particular sequences. That is not what PCR does.2755

It separates the DNA or protein fragments according to size.2766

Remember that PCR stands for polymerase chain reaction.2770

It amplifies a small piece of DNA during cycles of heating and cooling or manipulation of the genetic sequence of an organism.2775

Well, the purpose of PCR is to copy or amplify a target region, so that fits with C; so I am going to go ahead and put C here.2783

Electrophoresis: recall that electrophoresis involves putting DNA into wells in an agarose gel, applying an electric current to the gel2793

and then, letting that current do the work for you of separating out DNA fragments according to size2801

because smaller fragments will move more quickly towards the positive pole.2808

Cleave DNA, separates DNA or protein fragments according to size- that is correct, B.2815

Two left: restriction enzymes cleave DNA at specific sites in particular sequences- that is correct.2823

These were originally isolated from bacteria that would use the restriction enzymes to cut up DNA from invaders such as phage.2830

And now, we use them in the lab.2838

Finally, genetic engineering is the manipulation of the genetic sequence of an organism.2841

So PCR is C, gel electrophoresis, B; restriction enzyme is an A, and genetic engineering D.2849

Finally in example four, describe the phases involved in amplifying a segment of DNA using PCR.2858

Assuming you have all your materials together, you have the thermocycler machine,2865

the DNA you are trying to amplify, primers, nucleotides and TAC polymerase.2870

Then, you let the machine go through its cycle. The first stage is going to be the denaturing stage.2876

In this stage, the DNA is heated, and the hydrogen bonds between, and will break the DNA fragments will be single-stranded.2885

The next phase is the annealing phase. During this phase, the DNA is cooled, and that allows the primer to hydrogen bond with complementary DNA.2899

So, denaturing, this is phase one. Annealing is phase two.2915

And then, elong or extend phase, extend or elongate, this is, I am going to say, warm because it is heated up warmer than the annealing phase.2927

But it is not as hot at the denaturing phase.2940

TAC polymerase will use the nucleotides that we have added to elongate the primer.2943

So, denaturing, annealing and extending or elongating, those are the three major phases of PCR.2953

That concludes this lesson on biotechnology here at Educator.com.2961