Molecular Biology Basic Molecular Biology Research Techniques

Hi, and welcome back to www.educator.com.0000

Today’s lesson is going to be on the basic molecular biology research techniques0003

that you will probably come across in your lab session.0007

As an overview, we first have to talk about gel electrophoresis0012

because that is often going to be used in many different purposes for analyzing results.0014

From there, we will talk that restriction mapping going all the way through to DNA sequencing.0025

Gel Electrophoresis is a technique that is used to separate charged molecules, based only on their size.0033

It does this by using an electrical field that gets pulls through the gel, that has been submerged in a buffer solution.0043

Nucleic acids are an easy one because they are uniformly charged.0051

You have a constant charge to mass ratio.0057

The DNA will go from the negative pole down to the positive pole because DNA is negatively charged.0061

If we want to do gel electrophoresis on proteins which have a heterogeneous charge,0081

you have to coat them in a negatively charged dye so that they continue based solely on size.0088

The gel matrix allows for separation on size due to the pore size of the matrix.0102

We have two main gels that we would run.0107

We either have a polychromide matrix and that has a greater resolution than the other one which is called agarose.0110

Polychromide can get you down to the single base pair resolution.0119

You can tell the difference between a 25 base pair DNA and a 26 base pair DNA.0122

Where agarose is better when you want a 100 base pair resolution, something like that.0129

The topology will alter the rate of movement of the DNA molecule.0137

Nucleic acids first get linearized by being cut with restriction enzyme.0143

A relaxed circular piece of DNA will run slower than a linear piece of DNA.0149

A tightly wound supercoil DNA will run even faster than a linear DNA.0156

They take all of that guessing game out of it, you cut them with restriction enzymes or restriction endonucleases.0164

First things first, what we will do, we have a gel.0173

Let us say for example this is an agarose gel.0177

You would take your sample of DNA and usually what to do is you mix it with a colored dye, so you can see it running down the lane.0179

Not only that, you can see that you actually loaded it properly into the wells.0190

Those are wells that you make in the gel so that the molecules of the DNA0197

actually run through the gel matrix, instead of on top of it.0203

You usually always load a ladder in the leftmost well.0205

What a ladder is, it is actually a mixture of DNAs of different sizes but they are all known sizes.0211

It acts like a ruler.0220

For example, you would know that this is 100 base pairs, this is 200, this is 300, this is 400,0223

this is 500, and then maybe this is a thousand.0230

Then, you can see somewhere between here, if this is 100 and this is 200,0234

it looks like our samples are maybe at 175 base pairs.0239

What our gel might look like is, let us say this.0248

Here is our gel.0254

Well 1, 2, 3, 4, 5.0264

Well 1, it is going to have our ladder.0270

Well 2, let us say we have something that is right around here and then maybe we have something like this.0285

Well 3, we have something right here.0298

Well 4, we have something here, maybe something here.0302

First of all, what this is right here, we call this a smear.0311

A smear usually consists of many different molecules with differing sizes or many different molecules of differing super helical nature.0319

To understand that, let us talk about this.0336

If normally, if this would be your linear DNA, that is where we would expect this piece of DNA to run.0339

Over here, this is going to be your circular DNA.0350

It is not going to run as far down.0356

By the way, remember this is the negative pole, this is the positive pole,0359

and DNA is going to run down this way because DNA is negatively charged.0364

Your uncut circular DNA will hang out the highest.0371

Your linear DNA will run really where you expect it to be.0379

This is a 500 base pair marker and your DNA was 475 base pairs that run about where it should have run.0384

That is a nice one.0394

Over here, you would have a supercoiled piece of DNA, that is this.0396

Over here, this is an even more supercoiled DNA.0404

The more supercoiled you are, the further you run down the gel, the faster you run.0421

If we had two pieces of linear DNA, one that was longer, say this is a 600 base pair marker.0425

If this is 600 base pair, it will run in this area.0436

If you have another piece of linear DNA, let us say 100 base pairs, it will run in that area.0443

Linear basically runs at a good approximation, based on the ladder, whereas, anything with topology.0456

A relax or supercoiled circular DNA would not run at the proper rate.0465

The smear can be made up of many different versions of super helical DNA or if you had improper restriction cutting,0473

you can have many different linear fragments in the same range.0481

Onto those restriction endonucleases.0488

These are proteins or enzymes produced by bacteria.0492

They are used to protect against foreign DNA.0497

They recognize DNA that is trying to invade, such as maybe our bacteriophage λ DNA.0499

The restriction endonucleases will recognize that this is a foreign piece of DNA and it will try to just chop it up.0506

So that it can throw it away and cannot be infected by it.0512

These endonucleases are sequence specific DNA binding proteins.0516

They bind DNA as dimers and they recognize palindromic DNA sequences.0521

Meaning, it reads one way, the exact same as it reads backwards.0527

These will cleave DNA symmetrically on both strands of the DNA.0532

It can cleave either in a blunt fashion or getting sticky ends.0536

I will show you what that means on the next slide.0541

The blunts or overhanging sticky ends.0545

If for example, we were to cleave right here.0549

There is a cleavage right there, that would be blunt because we have a GAA, CTT, that is cut.0555

And then, we have a TTC, AAG, that is blunt.0565

If it cuts, as we see right here, we will have overhanging ends or sticky ends.0570

It cuts at G, we separate, this is a CTTAA.0576

This right here is an AATC.0588

This is the sticky ends, we have an overhang both right here and right here.0598

We have complementary base pairing by the sticky ends.0608

These will want to come back together eventually and ligate, because look,0611

there is complementarity between the two strands.0616

This is an important aspect of restriction enzymes that we have been able to utilize for biotechnology.0620

I will tell you about that in just a couple of slides.0628

The length of the recognition sequence will determine the approximate cleavage frequency,0630

meaning right here, this is E. Coli 1 restriction enzyme.0635

It recognizes a 6 base pair sequence.0639

If we have another enzyme that recognizes a 4 base pair sequence,0644

the probability is that it will cut more frequently throughout the genome0650

because it is only looking for every 4 sequence, instead of every 6.0654

Basically, if you want to cut up a genome very frequently,0660

you use a restriction enzyme with that has maybe only 4 bases recognition sequence.0664

If you want to cut it up less frequently, you would use maybe a 6 base pair recognition sequence such as one.0670

Another technique that we are going to see a lot in molecular biology is called cloning.0682

What cloning is, is we are using what is called a plasmid vector.0689

This is a piece of DNA that contains a replication origin, as well as, usually,0695

an antibiotic resistance marker, and then your piece of DNA, after it is all said and done.0704

We have this replication origin.0712

The reason we need that is because every time, once we put this into an E. Coli or any bacteria,0714

every time the E. Coli replicates, the plasmid vector will replicate.0721

And then, we have a selection marker usually as antibiotic resistance.0727

We have a DNA fragment that we want to look at.0732

We do not know what the sequence is.0735

We cut it with a restriction enzyme.0737

We also cut the vector with the exact same restriction enzyme so they have complementary sticky ends.0740

We then mix those together, use DNA ligase to ligate them together.0747

And then, we transform them into bacteria meaning we put that vector into a bacterium.0753

And then, we allow that bacteria to grow so that we make more copies of the vector.0759

And then, we plate them on a media that has whatever antibiotic.0765

If we have an antibiotic resistance for ampicillin, then we plate them on ampicillin plates.0772

Therefore, only the bacteria that received the vector will be able to grow on that ampicillin plates.0782

Anywhere where that colony is, you can know for sure that it picked up a vector.0791

How does this work, let us write this up.0800

We have our vector.0804

It has antibiotic resistance marker right here.0809

It also has an origin of replication.0819

This is our vector.0826

We are going to cut this with an endonuclease restriction enzyme.0830

What that is going to give us is this.0839

We still have our, let us say ampicillin resistance and this gives us some sticky ends.0845

This now has sticky ends or overhanging ends due to the cut with the specific endonuclease that we used.0852

We are going to take our DNA insert, our DNA of interest, and maybe we do not even know what the sequence is.0861

We are also going to cut it.0870

We are going to cut this with the exact same endonuclease that we cut our vector with.0877

That DNA is going to have sticky ends.0885

Maybe it has overhangs on both sides, right there.0892

We have sticky ends.0894

What we can do is we can add these together in solution with DNA ligase.0898

What we would get would be, we still have our origin.0913

We have our ampicillin antibiotic resistance.0925

Now we have our DNA insert in there.0930

Because of the complementarity between the sticky ends,0936

the ligase was able to just put those two together like it was always there.0939

From here, we can add into E. Coli, this E. Coli with its circular genome.0945

If we go through transformation, usually this is done by just heat shocking the bacterium at about 37° C.0966

What they does is it opens up pores in the bacterial cell wall which allows the plasmid vector to come in.0980

Now, what we have, in size relation, it is much smaller than the bacterial genome.0992

We have here a plasmid coming in, still has its insert, it has its origin, it has its antibiotic resistance.0999

And then, we allow the E. Coli to recover.1011

All of the holes in the membrane come back up.1018

This is E. Coli.1023

From there, we can grow this up in solution so that every time it replicates about every 30 minutes,1027

it will not only replicate the DNA genome but it will also replicate the plasmid.1034

We grow that up over and over.1040

We can do that in a test tube.1044

From there, we can plate it on our antibiotic resistant plates.1048

What we are going to see is that we are going to find colonies.1064

Each one of these colonies was made from an individual bacteria.1071

Each one of these colonies on an ampicillin plate, with these being ampicillin resistant,1075

each one of these that grow on their had to have had this vector with the ampicillin resistance.1085

You can then pick these up from there, grow up a bunch of them.1092

You would say, I will pick this one and grow it in there.1101

Pick that one, grow in there.1108

You grow them all up, you can purify the plasmid out.1110

And then, you can either do a PCR which we will talk about later or you can do DNA sequencing which we will also talk about later,1116

to confirm that not only did this colony have a vector but it had the vector that actually had the insert in it.1126

That is a big difference.1136

Just because it grew on this plate, all we know is that it had the ampicillin resistance, so it had the vector.1137

We cannot be sure that it had the insert, unless we do a second check.1143

From here, we would purify that DNA and then check for the insert either by PCR or by DNA sequencing.1148

From there, if we do want that DNA piece of interest, maybe it is a protein coding genome,1159

maybe we can grow up a bunch of protein.1166

Maybe, we just wanted a bunch more DNA, there is a whole bunch of things we can do with it.1168

To switch gears just a little bit but be able to use all of what we have seen so far,1176

by using cloning, by using transformation, by utilizing restriction enzymes, we can create a genomic DNA library.1182

We call this library prep.1190

What you do is you are isolating the entire genomic DNA of an organism.1192

You are going to break it down into usable sizes.1199

You cut the DNA to appropriate sizes using your restriction enzymes and you ligate it into a vector.1204

And then, what you are going to do is through clotting.1211

And then, you are going to take that cloned plasmid vector.1215

You are going to transform it into bacteria.1219

You are going to grow those up on the antibiotic media like we talked about.1222

Because each plasmid that we can make, we will have a different DNA insert containing different genes,1228

the total collection of everything altogether, your entire library will represent the whole genome.1236

From there, you can do sequencing or you can do protein preparations.1242

You can do a whole bunch of things.1246

Oftentimes, we are doing DNA sequencing to find the sequence of that entire genome.1247

I have talked about PCR using polymerase chain reaction to confirm your insert.1256

Polymerase chain reaction is an extremely useful and extremely vital molecular biology technique.1263

It is used to amplify a small amount of DNA molecules to millions, hundreds of millions, in maybe 2 or 3 hours.1272

Luckily now, it is an automated process.1281

It does not require the use of bacteria unlike cloning does or transformation,1285

because you are copying this DNA completely separate from a living organism.1293

What I want to point out right here is this is the protein which is called taq polymerase.1300

Taq polymerase is a thermostable polymerase meaning it can withstand high temperatures.1316

It was actually found in a bacterium that is what is called extremophile or thermophile.1324

It lives in hot vents, hot springs.1331

This is what is called a thermocycler.1337

This machine allows PCR to be completed in an automated process.1345

What does PCR require?1354

You need template DNA, something that you want to amplify.1357

You need a primer parent that is complementary to the DNA sequences that are outside of that room.1363

First, for example, this is your DNA region of interest.1371

The black is DNA that we know the sequence of.1378

You would make you primer specific for the sequence that you know.1382

What it would do is, as you go through the synthesis, you will make many copies of your unknowns sequent.1387

From there, you can actually do many things with it to check what is that sequence is.1396

You also need a thermostable DNA polymerase, like we talked about, our taq.1402

You need DNTPS which is our deoxy nucleoside triphosphate, our ATPs, GTPs, ATG and CTPs, the deoxy form.1407

What we are going to do over and over again are 3 steps.1420

First, we are going to denature.1423

We are going to pull the strands apart by melting the hydrogen bonds, at 95° C.1425

Next, we are going to anneal the primers, at anywhere between 50 and 65° C so that allows those hydrogen bonds to occur.1432

And then, we are going to extend by increasing the temperature,1447

in this case it is 70° C because that is where taq polymerase works the best.1451

A little story first before we go on, is that when PCR was originally developed in the 70’s,1457

what we had is that we had 3 different water baths being set up.1470

One was set up at 95, one was set up at roughly 60, and one was set up at lower than 72.1485

I cannot remember what is the exact number was.1495

Let us just say this was 50 and let us say this was 60° C.1499

What you had is a single tube with your reaction mixture in it, being held in the 95° water bath for about 10 seconds.1504

Then, you would move to the 50° C water bath.1518

At this point, you would have to add in DNA polymerase.1523

Then you would hold it in the 50° C water bath for maybe 30 seconds,1532

then you would hold in the 60° water bath for maybe a minute.1537

Then, you would repeat, that would be one cycle.1547

Then, you go to the 95°, put in there for 10 seconds or so.1549

At the 95°, you would kill the DNA polymerase because it was not heat stable.1555

Every time after the 95° C, you would have to add in DNA polymerase every time.1560

That is why you had to do it by hand.1568

The discovery of the thermostable DNA polymerase, taq polymerase,1571

allowed you to not have to add in polymerase every single time you went through the denaturing step.1576

Therefore, that was actually the most important discovery for allowing this process to become automated.1582

That is a huge discovery.1591

PCR, absolutely huge invention.1592

Taq polymerase, absolutely huge for mechanizing this technique.1596

Let us talk about the technique, PCR, what is it look like?1602

First of all, let us go with our DNA sequence.1607

Here is our DNA sequence.1616

This is our sequence of interest, let us say.1619

Obviously this is hydrogen bonded at this point.1624

What we are going to do, we are going to put it 95° C to denature.1632

What is that going to give us, that is going to give us completely separate strands.1644

Once again, this is our region of interest.1651

Next thing we are going to do is, we are going to put it from anywhere from 50-60° C.1658

This is for annealing of the primers.1668

These are primers that are going to be specific to just outside our region of interest,1675

because maybe we do not know what that region of interest is, at this point.1679

Here is our region of interest, once again.1690

I will write my primers in different colors.1695

Let us say this one.1703

That is why it looked a little weird on my paper.1765

This would be the 3 prime, 5 prime, 3 prime, 5 prime, 3 prime, 5 prime, 3 prime.1770

That makes a lot more sense.1783

Here is our primer there, here we have our primer here with its 5 prime, that is the annealing.1786

The next step is our elongation.1794

This is at 72° C, we have elongation.1800

That is when we have taq polymerase coming in and lengthening, synthesizing DNA.1806

We have 5 prime, another strand 3 prime.1818

DNA of interest, we have our primer.1827

Elongation is going to continue in a 5 prime to 3 prime fashion, going all the way through.1837

Over here, 5 prime to 3 prime fashion, all the way through, perfect.1844

If we go to the next step, we are going to go denature again.1858

95, we are going to denature.1865

What we have here is 5 prime.1869

This is our original one.1882

We have this which has the sequence of interest.1890

We also have this one, the sequence of interest.1908

And then we have this one, with the sequence of interest.1918

What you would do again is you would go through annealing of the primers all over again.1931

What you end up getting is you would now make, the anneal primer.1940

Let us say here again.1947

That should be purple.1951

Go that way during elongation.1958

You would anneal a primer here going that way during elongation.1962

That would actually be this.1975

This one, it would be here and here, going through elongation and then denature again.1982

You denature, anneal, extend, denature, anneal, extend, over and over again.2002

What you end up having is after your third round, you finally have your sequence of interest double stranded,2008

completely separated from any other excess DNAs.2026

If you can see here, we are going to have excess DNA in the leftward direction.2033

If you see here, excess DNA in the rightward direction.2038

Even after here, we are going to have some excess DNA in either direction.2040

After the 3rd round, you finally have just your sequence of interest.2045

After every cycle after that, this is going to dominate in your reaction mixture2050

because it is so much smaller and so much more specific.2055

It will just replicate over and over again, you will get millions and millions of copies of this.2058

In which case, you can isolate that and utilize it for whatever your next part of your experiment is for,2063

whether it would be DNA sequencing, whether it be inserting this as your insert into a plasmid vector for cloning,2073

so many different things.2081

What can you do with PCR is, as I said you can do cloning.2087

You can catch criminals, you do the DNA tests to see if somebody murdered.2090

Or you can see somebody whose paternity test, who is the father.2097

You can do genome sequencing with PCR.2102

You can do mutagenesis, you can make things for future experiments like making probes.2104

You can even do gene expression profoundly, when you talk about doing PCR and amplifying regions originally from mRNA.2111

There are many things you can do with PCR.2123

Let us move on to a different technique which is called the southern blot.2127

This was actually invented by a man Dr. Southern and that is why it is named southern blot.2133

It is a technique used for detection of DNA, a specific DNA sequence.2144

What happens here is you use restriction endonucleases.2150

What we talked about before.2154

They cut high molecular weight DNA into smaller pieces, so there are more easily manageable.2156

Those DNA fragments are separated using that agarose gel electrophoresis.2161

They get denatured, making sure that they are single stranded.2167

The DNA gets transferred from a gel to a nitro cellulose membrane.2171

The reason they do this is because agarose gel, it has a consistency of a jello.2175

It does not have a long shelf life or maybe you can break it.2181

Once you can put it on a nitro cellulose membrane, it is much easier for longevity purposes.2187

Once you have, you basically make a complete stamp, whatever is on the gel,2198

you transfer that into a nitro cellulose membrane by using a charge in a buffer, the electrical current through a buffer.2203

And then, on membrane, you can treat it with a radioactive DNA probe that is complementary for your DNA sequence of interest.2211

The probe will only show up where your sequence of interest shows up.2220

You can detect that via on Xray film.2227

Another type of blot is a western blot.2234

This is a technique that detects proteins of interest.2237

You can either use purified protein or you can use just a cell lysate.2241

What that is, is take a cell, you break it up and everything in there is just ran into the gel.2244

In this case, an STS page gel, that is a type of polychromide gel.2252

The STS is a detergent, it denatures the proteins so you are allowed to run based on size only and not any type of confirmation.2259

The proteins get transferred from the gel to a nitro cellulose membrane, same reasoning.2269

It is more easily transferable and longevity.2274

The membrane gets incubator with the milk blocking solution because there are holes in the membrane, there are pores.2279

You do not want antibodies, which we are going to add later to be stuck into the pores and cause nonspecific binding.2287

You coat it with milk proteins then you add primary antibody.2296

This is an antibody that specific for your protein element.2301

Then, you add a secondary antibody that is specific for that primary antibody.2306

The reason you do this is because it allows for amplification of your signal.2310

You can do the same process without the secondary antibody.2315

But you get a lot lower yield and it is harder to see the bands on this gel.2320

Then, you can finally detect the protein via either colorimetric or luminescence.2327

Colorimetric is you can physically see it with your own eyes.2333

Maybe it is like purplish or gray on the nitro cellulose membrane, where your protein of interest is.2337

Luminescence, you use xray film to develop it.2343

A northern blot is a technique that detects RNA sequence of interest.2348

Remember, southern blot detects DNA.2353

Western blot, protein.2356

Northern blot, RNA.2358

You can use a northern blot to analyze mRNA.2361

That analysis would reveal expression levels of different genes.2365

As we talked about before, just because you have high or low mRNA expression2371

does not necessarily mean that translates over to the protein expression but sometimes it is linked.2376

RNA, you run it on agarose gel to separate via size.2386

You do it on denaturing condition so you do not have any secondary structures.2391

After separation of RNA, you transfer it to a membrane, longevity.2396

And then, you add a labeled probe that is complementary to the RNA.2401

This is very similar to the DNA blot, the southern blot.2407

To look like what all of these might seem like, you have your sample, this is specifically for northern blot.2412

You have your sample, you extract your RNA.2418

You go through electrophoresis to separate based on size.2421

You transfer to a membrane for longevity.2425

From there, you add labeled probes that will only bind to your protein of interest.2430

From there, in this case, these are labeled probes that you can see by putting an xray film under the radioactive.2436

Only where the probe show up will you see bands on this xray film.2447

Only where your protein of interest shows up, will the probes have showed up because it is a complementary sequence.2452

This shows you your protein.2460

This is very nice compared to, say an agarose gel where there are many bands and you do not know which one you protein is.2464

This is a way to isolate exactly which one your protein is.2471

Moving on to another technique.2482

We can make the DNA from RNA.2483

This is what we called cDNA synthesis or complementary DNA synthesis.2488

We can convert an mRNA into DNA.2494

This is very important because this allows us to look at our gene expression.2499

These are usually our protein coding genes, the mRNAs.2503

This, even though MRNA is less than 5% of our total RNA, it is the only one that gets put in a protein.2509

This, what we think is very interesting.2515

The good, very good thing, the very unique thing about mRNA compared to rRNA, tRNA, and DNA,2518

is it has a poly-A tail at the 3 prime.2527

We can utilize that feature to isolate MRNA from any other nucleic acid.2531

We do that by using an oligo DT primer meaning we have many Ts.2539

What are those T's complementary to?2547

All of those A’s that we find in the poly-A tail.2550

We can use that to bind to our poly-A tail.2554

We can pull that out of solution and all the rest of the RNAs that do not have this poly-A tail will be washed away.2558

Oligo DT is used to prime DNA synthesis then using an enzyme called reverse transcriptase.2568

We are turning RNA into DNA.2577

The central dogma goes down, but reverse transcriptase enzyme violates that and turns RNA into DNA.2587

MRNA uses a temple to make a complementary DNA strand.2595

Now, we have our mRNA in blue.2600

We have our complementary DNA in red.2608

Then, we can degrade our mRNA and then synthesize a new piece of DNA complementary to that cDNA.2611

Now, we have a full double stranded piece of DNA, in which case we can make many copies of utilizing PCR.2624

The cDNA strand is copied by DNA polymerase to make double stranded DNA, like I said right here.2633

Since this came from mature mRNA, all the intron should have been spliced out.2639

Now, we only have the exonic region.2643

We only have the protein coding region which is really cool.2646

A cool thing that we can do once we have made the cDNA is that we can quantify it.2655

We can do that using quantitative PCR, quantitative polymerase chain reaction, otherwise known as qpcr.2660

This is a technique to quantify the amount of cDNA.2668

Therefore, indirectly all the mRNA transcript of the specific sequent.2671

Of this gene that we are looking at, gene X, we can see how many mRNA transcripts are made of this gene.2678

Maybe, we can hope that it is fairly, directly correlated with how much protein is made.2688

This is a measure of our gene expression.2695

It is much more sensitive than the gene expression quantitation we can get from northern blot.2699

But you are still only getting one gene analysis at a time.2705

Although, you can do QPCR in plates that have 96 or 384 wells, meaning you can have a different reaction in every well.2710

Right down here, this is what you would see or read out of a QPCR machine.2722

What you are saying is you can compare that, say this is your control.2727

What we are saying is anything that crosses this boundary to the left of the control2738

means it has an increased gene expression, more mRNA transcripts.2748

Anything to the right, you have decreased gene expression, less mRNA transcripts were found.2756

You can do that based on, maybe a drug treatment, maybe interaction with certain protein.2763

Maybe knockout a gene or add a gene somewhere else.2768

You can see how that affects other genes.2770

You can do this in a much more high throughput way, by looking at the transcriptome via microarrays.2776

The transcriptome is just a collection of all of our transcription in the cells.2783

We are usually referring to mRNAs because these are things that can be made into proteins.2789

We use microarrays and these are just very small slides or chips that contain a bunch of DNA that is attached to there.2795

Each of these probes will correspond to a different gene.2807

We do our cDNA synthesis from our mRNA samples.2812

What we do is that we have our cDNA from our experimental mRNA sample, being labeled fluorescently with a green tag.2816

Maybe our control mRNA sample with a red tag.2826

You hybridized both of those at the same time.2831

You allow them to bind the DNA chip, at the same time to the same chip.2833

For each spot on this chip, right here that we are looking at, each spot represents a different gene.2842

The color on this chip after hybridization of the control sample and experimental sample,2851

will indicate whether the mRNA was more abundant for the control or the experiment.2858

For example, if we see a green sample right here, this is saying that we had more transcripts of our experimental sample.2864

We had an increase gene expression.2881

If we see a red over here, we have much more of our control sample.2884

That means, whatever treatment, whatever that experiment did, down regulated that mRNA transcript.2893

If you see over here like the yellow one, that is saying that there is no change.2899

Your experiment and your control have this roughly same amount of mRNA.2908

This can tell you for particular condition, whether a gene is up regulated or down regulated compared to the control.2915

All genes can be analyzed simultaneously, based on however many genes can be printed on a single chip.2923

For an organism like yeast, you can get all of the genes on a single chip.2930

For humans, we would not be able to get all the genes on a single chip.2939

This is going to be less quantitative than QPCR and northern blot because you can just see that it is red or it is yellow, or it is green.2945

You cannot really tell how much more you have up regulated or down regulated.2955

You can just tell that you have.2961

But we can utilize another type of technique called RNA sequencing or RNAseq that is used from our cDNA library sequencing,2964

being much higher throughput and much more quantitative2975

because you can do the entire genome at one time and you can get actual quantity.2978

You can get number saying that the experimental sample had 1.25 times more transcripts mRNA than in the control sample.2984

RNAseq is a very highly utilized technique nowadays for gene expression.3000

Talking about RNA sequencing, that is going to bring this to DNA sequencing.3012

Let us explain DNA sequencing first.3018

Let us start with Sanger sequencing which was developed by Frederick Sanger.3022

He won a noble prize for this, it was actually his second Nobel prize.3026

What he utilized was a tool of dideoxynucleotides.3030

Dideoxynucleotides lack both a 2 prime hydroxyl and a 3 prime hydroxyl.3036

The 2 prime hydroxyl lacking means that this is a deoxyribose, this is a DNA base.3042

The lack of the 3 prime hydroxyl does not allow a new base to be added in because remember, we add 5 prime to 3 prime.3050

The next incoming base would have a 5 prime phosphate, would normally add right there.3061

That 5 prime phosphate, if we want to add, it cannot make that phospodiester bond3073

because there is no alcohol group for that ester bond to be made.3082

Effectively, it terminates the chain.3088

Whenever dideoxynucleotide triphosphate is added in, that is the last nucleotide added in that particular chain.3090

We call it a chain terminator.3100

Incorporation of DDNTP blocks further polymerization.3103

In DNA sequencing, we have a primer being annealed to a DNA region of interest.3112

We are going to basically utilize the PCR steps.3117

The primer, in the presence of all 4 normal dntps, as well as DNA polymerase,3120

the primary gets extended to the end of the template.3129

Right here is the annealing phase, this is the elongation phase of PCR.3132

In the additional presence of a small proportion of our DDNTPS, usually about maybe 10 to 1 normal, DNTP vs. DDNTPS.3138

Every once in a while, a DDNTP gets incorporated and that will terminate the chain.3158

The position of that DDNTP will indicate the position of its complementary base on the template DNA.3163

If let us say a DDGTP was incorporated into this growing chain, that would show that on the template DNA that was actually a DCTP.3169

Because we know the G’s and C's make a complementary bond.3183

Originally, how you could analyze this was by running a polychromide gel, a polychromide gel electrophoresis.3197

Our sequencing gel is run, where you had 4 different reactions being performed, each with a different DDNTP.3206

Each reaction was run in a lane on a denaturing polyacrylamide gel3215

which allow the analysis of the single synthesize strand with single base pair resolution.3220

That is why they are using polyacrylamide gel instead of the agarose.3224

The position of the band in the lane indicates where each base was incorporated, that make sense.3229

How would we read one of these?3235

Let us draw this out.3237

This was back when it was first done.3250

We do not use sequencing gels as much anymore.3252

We would normally just use a automated sequencer.3256

We will do our wells.3261

This is our ladder.3270

We will say that is 1 and this is 10.3290

In this we will say that we added our normal DNTPS, as well as, this is DDATP.3297

This one was DDGTP, this one is DDCTP, this is DDTTP.3310

Originally, only a single dideoxynucleotide was added in one time.3325

What you would get is something like this.3331

This is what would be a readout, this is what it would look like.3368

How you would read this is as such.3373

You come down here, obviously the ones that are the smallest run the further.3375

You look down here and you look at the first nucleotide.3380

You find that was found in the DDATP.3383

The DDATP is saying that this was found at the first nucleotide meaning that it is complementary to a T in the sequence.3389

If we write this out from 5 prime to 3 prime, what our sequence is, we are going to start with here, we found a DDATP.3399

That is going to be a T.3406

Our second one, we found the DDGTP was found here, so that is going to be a C.3410

Where is the third, 3rd and 4th are found in the DDTTP lane, those are going to be a's.3420

The 5th one, DDGTP, it is a C.3430

6th, the T, so an A.3434

7th, CTP that is a G.3437

8th, the ATP it is a T.3441

9th and 10th are the CTPs, those are G.3444

This would be the sequence of a 10 base template DNA sequence.3449

This would be our template DNA.3455

This is the sequence and this was unknown at the time, we did not know it.3460

But we figured out by doing DNA sequencing.3464

This can be done in much more high throughput ways.3468

Now, we can actually do dideoxy sequencing reactions being carried out in large batches.3472

Maybe even up to 384 reactions at a time, inside machines.3477

These machines can separate samples in capillary tubes.3484

Each one of those capillary tubes, very thin glass tubes, are filled with a matrix of polyacrylamide gel.3489

It is basically like running 384 individual gels but it is all machine operated.3496

The sequencing reactions are carried out altogether in a single reaction, in a single test tube.3505

You would have your normal DNTPS and you would have all of your DDNTPS or DDATP, DDTTP, DDGTP, and DDCTP, all in the same reaction.3514

How they can do that is that each DDNTP is conjugated to a different plates and dye.3534

Maybe A is conjugated to a red dye.3541

T is conjugated into a purple dye, G a green dye and C maybe a blue dye.3546

As the molecules would travel through the gel in the capillary, they get separated based on size because the smaller ones travel faster.3561

The DNA molecules, as they reach the end of the capillaries will cross a laser light.3570

This laser light will excite the fluorescent dyes attached to each DDNTP.3579

There is a camera, a CCD camera, close capture device camera,3585

that captures the wavelength of light and records the color accordingly.3590

The sequence of DNA then will be displayed as what is called a trace, shown below.3594

As we can see, as the 1st DNA base comes through, it is red.3600

In red, that fluorescent dye is associated with a T.3608

The next one that comes is blue, that one is associated with a C.3614

The 3rd one green that is associated with an A, so and so forth,3618

all the way through until you finished the entire length of the synthesized DNA molecule.3622

Usually, it is no more than about a thousand bases long.3631

You can this many times.3635

What you do is you do it for all the pieces of the genome.3639

You sequence that genome hopefully the same part of the genome 5 or 10 times.3644

That is what they call 5x coverage or 10x coverage.3650

You do that with a large amount of reads.3653

The reason you that is because, let us say you want 10x coverage.3659

The reason being, let us start with 5, maybe for this piece of DNA, 5 pieces,3663

you have an A in this spot for 4 of them but you have a T in this spot for one piece of that DNA.3673

You are going to go majority rules and you are going to call this an A.3684

Every once in a while there are single nucleotide polymorphisms between DNA and species.3690

For making the reference genome, you take just the majority rules type of approach.3700

Once you make a reference genome then you can compare individual genomes and look for these specific differences.3707

But when you are at the first part making your reference genome, majority rules.3716

Once you get all these sequences, they get entered into the database and now the rest of the stuff is computational.3722

You search for overlapping sequences.3728

You are looking at tiling arrays, it is really just a big game of puzzle making.3731

What you get is you order overlapping sequences into what are called contiguous sequences or contigs.3738

This gets repeated over and over again, as you can see here, we know the sequence and3746

we just make overlapping reads until we can fill out the entire sequence of the genome.3751

Once you get that first genome made which is called the reference genome, it makes it much easier3762

because you can have missing pieces or you can have pieces that you do not quite know where they map to.3769

This one, you may not know where it goes but when you have a reference genome sequence,3777

you can say okay it does not go over here, it goes right here.3780

It is like making a puzzle with out the picture on a box, that is when you are making the first genome,3786

the first reference genome sequent.3793

When you do have the picture on the box, you know what the puzzle supposed to look like that is when you have a reference genome.3795

This is what, from start to finish, the genome sequencing might look like.3806

You make your reaction mixture, your PCR mixture, with your DDNTPS.3811

You go through the Sanger sequencing synthesis process, making your DNA sequences of many different lengths.3816

Because at any random time, you are likely to get a chain terminator to stop in there.3831

You stop at one base pair, at two base pair, at 3, at 4, 5, all the way through.3838

From there, you load it into a DNA sequencer that will run through a capillary.3843

As you run toward the end of the capillary, you pass through a laser, that laser excites the dye on the dideoxynucleotide.3850

That excitation wavelength is captured by a CCD detector.3860

From there, you can do the laser detection and sequence analysis.3866

You get a chromatogram and you can say that okay this was an A, that was a T, that was a T,3872

that was an A, this was a C, that was a G, so on and so forth.3877

This is really important and this has allowed the human genome sequence to be completed,3882

as well as all of the genome sequencing.3889

We now can do next generation sequencing which does not use capillary sequencing.3893

We can get many more read, much more sequence, instead of maybe 300,000,000 bases at a time.3898

Maybe we can get 3 billion bases at a time.3909

In theory, we could sequence an entire human genome in one run, our next generation sequencing,3913

instead of several runs on the old capillary gel sequencing.3921

My last slide, I want to leave you with is just an example, just talking about the human genome project.3927

It was a huge effort started by the government to sequence the human genome.3933

It was started in 1990 and fully completed in 2003.3941

The first human genome sequence was published in February 2001.3946

It started as a government funded and government ran sequencing project.3952

This man right here, his name is J. Craig Venter.3961

Without this man in Celera Genomics at the time, we might still be waiting for the human genome to be completed.3971

Under his guidance, he pushed for shotgun sequencing.3981

Shotgun sequencing is a way that was not used by the government at the time.3988

Their sequencing methods were extremely slow.3999

Shotgun sequencing is a technique to basically take your extracted DNA, shotgun the base,4003

you blow it up into a bunch of different sequences.4010

Clone those vectors, grow them up in plasmids, and then sequence the DNA library.4013

From there, you take all your different sequences, you tile them together, and get your full genome sequencing.4020

J. Craig Venter really pushed shotgun sequencing.4030

This was a technique that was not looked highly upon, by many scientists at the time.4033

It was often thought of as more error prone, less sensitive to anything.4038

Without his Celera Genomics foray into the human genome project using private funded money,4047

we may not have gotten the human genome.4056

We definitely would not have gotten its sequences, as early as we did, and we may still be working on it.4060

Just getting the human genome project finished, we have been able to have many new novel discoveries,4066

based upon different genomes being compared to the reference genomes, using the human genome project.4074

We can look at genetic defects, we can look at cancer genes, we can look at DNA mutations affecting maybe DNA repair or development.4081

The human genome project was a huge effort.4093

In my opinion, one of the biggest successes in biotechnology of our generation.4102

I hope you enjoyed this lecture, this is the end of it so far.4111

I will see you next week, hopefully.4115

Thank you so much for joining us at www.educator.com, and I hope to see you again.4118