Chapter 10 material - Weber State Universityfaculty.weber.edu/btrask/Chapter_10_material.pdf ·...

Chapter 10

Analyzing Genes

and Genomes

Copyright © Garland Science 2010

These slides were made to provide direction when

reading Chapter 10 in Essential Cell Biology—please

read the sections corresponding to the figures in this ppt

so that you thoroughly understand the concepts. I would

like you to be very familiar with the techniques in this

ppt, particularly the techniques that are underlined.

We’ll spend time in class next week to answer any

questions you might have.

--Barb

Restriction Enzymes(Examples on next slide)

• Cut DNA at defined sites (recognition site has to be exact)

• Isolated from bacteria

� Restriction enzymes (REs) are named after the bacterium from which they were

isolated

� Ex: EcoRI is from E. coli, strain R, and the 1st enzyme isolated from that strain; Hind

III is from Haemophilus influenza, strain d, and the 3rd enzyme isolated from that strain

• Bacteria produce these as ‘innate immune’ molecules—to cut any DNA from

foreign invaders (note: bacteria that produce a given RE cannot have the

recognition site in their own genomes)

• We can buy isolated REs and use them to cut DNA of ANY species that has a

sequence that is recognized. We call this, “restriction digestion.”

• If a recognition sequence is 4 nts long, the likelihood of it appearing in any DNA

(assuming random sequence) is 4x4x4x4=256 nts, and a 6 nt long recognition

sequence is likely to occur once every 4x4x4x4x4x4= every 4096 nts

Figure 10-2 Essential Cell Biology (© Garland Science 2010)

Ligation

• After DNA is cut with an RE, it often has ‘overhanging’

ends—any DNA (from ANY species) cut with the same RE

will have the same overhanging ends.

• If the DNA is mixed, the overhanging ends will be

complementary and will hydrogen bond with any fragment of

DNA with a complementary end.

• If the enzyme DNA ligase is added to the mixture, the nicks in

the DNA backbone can be sealed. In this way, 2 fragments of

DNA, potentially from different species, can be ‘glued’

together to make a “recombinant DNA” molecule.

• See the example on the next slide.

Figure 10-6a Essential Cell Biology (© Garland Science 2010)

RESTRICTION DIGESTION WITH EcoRI

Ligation

Reaction

If we cut DNA with an RE, we can separate

the fragments by gel electrophoresis.

mRNA

These figures to

the left show

separation of

mRNA, but any

nucleic acid can

be separated in

this manner,

including DNA

‘Northern blotting’ is a technique used to determine whether a

specific RNA sequence is present in a given sample (tissue, cell,

organ, etc.)

• RNAs are isolated, separated by electrophoresis (by size), and

transferred to a solid support (nitrocellulose)

• The nitrocellulose ‘copy’ of the gel is incubated with a labeled

(radioactive or color) “probe”; a “probe” is a DNA sequence that is

complementary to the RNA

• The labeled DNA probe binds to its complementary RNA (assuming

it’s there) and because it is labeled, identifies whether your RNA of

interest is present in the sample

Figure 10-5 (part 1 of 5) Essential Cell Biology (© Garland Science 2010)

Recombinant DNA can be ‘Cloned’

• Molecular cloning occurs when a molecule of DNA is ‘amplified’ (make

many copies)

• If we make recombinant DNA by ligating a fragment of interest into a

bacterial plasmid (a non-chromosomal circular DNA that are isolated

from bacteria), we can re-insert the modified plasmid into bacteria.

� Once in the bacteria, the recombinant plasmid will amplify with the bacteria

as they live/grow, so we can obtain many copies of our DNA fragment of

interest (if we re-isolate it from the bacteria.

• See the next slides

Reverse Transcription

• This is something we (eukaryotic cells) don’t do—even

prokaryotes don’t do this. BUT, some viruses are able to

convert RNA � DNA via reverse transcription, using a

reverse transcriptase enzyme.

• We can use this enzyme in the lab to convert RNA back to

DNA.

� This is especially useful if we convert mRNA into DNA

because we can obtain a DNA copy (called ‘cDNA’) of the mRNA—

which represents only the exonic parts of a gene.

• See the next slide

• After converting RNA � DNA via reverse transcription,

we can get billions of copies of the DNA by subjecting it

to polymerase chain reaction (PCR).

• The coupling of reverse transcription with polymerase

chain reaction is known as RT-PCR.

• The end result is billions of copies of a cDNA—DNA

copies of an mRNA.

• See the next slides

PCR and RT-PCR


PCR

PCR is basically an in vitro DNA polymerase reaction. We have a template, a DNA

polymerase enzyme, buffers, nucleotides, and two primers and we simply let the

polymerase reaction proceed.

There are a few things that are different about PCR and a regular polymerase reaction.

Mostly, our in vitro tube does not have all of the other enzymes (e.g., helicase, initiator

proteins) that are in a cell, so we have to force the DNA apart by heating it. In addition,

we do the reaction multiple times in tandem.

PCR is a three-step process that is repeated multiple times in tandem. The three steps

are: 1.) heating the DNA to separate the two strands of the template, 2.) lowering the

temperature so primers can bind, 3.) allowing the polymerase to copy the template.

Note that we have to include primers because the polymerase enzyme can’t start

polymerizing but needs to add nucleotides onto a primer. The primers used in PCR are

DNA primers—not like the RNA primers that are used in vivo. And we nee to include

TWO primers because we want to copy both strands.

See the next slide.

These three steps are repeated over and over again in tandem to generate numerous copies of

the original DNA template.

Because the heating step is repeated numerous times, we need a heat-stable polymerase

enzyme—one that can withstand the repeated heating steps. The polymerase we use is isolated

from a bacterium that lives in thermal hot springs: Thermus aquaticus. Thus, we call the heat-

stable DNA polymerase “Taq polymerase.”


This figure shows just three cycles. We generally do at least 30, giving us billions

of copies of double stranded DNA—the DNA that exists between the two primers

that we’ve used.


Comparison of

PCR to RT-

PCR


Another use of RT-PCRIt’s a way to detect retroviral infection easily by

collecting just a little bit of blood from a potentially

infected individual.

Figure 10-19a Essential Cell Biology (© Garland Science 2010)

A use of PCRLook at the size of a specific DNA area (called a DNA

‘locus’; different individuals can have alternative

‘polymorphisms’ in their DNA

Figure 10-19b Essential Cell Biology (© Garland Science 2010)

Same thing as the previous

slide, but for several different

gene loci—if many loci are

included in a genetic

comparison, we can

potentially [if enough loci

are analyzed] whether two

DNA samples are from the

same individual (e.g., if the

blood at the crime matches

YOUR blood!)

DNA Sequencing

It’s often necessary to know a specific sequence of nucleotides—

for example, if we need to make probes for northern blotting or

primers for PCR, we need to know those sequences.

The method used to determine a DNA’s sequence is the same that

has been used for several decades; it was developed by a researcher

names Sanger, so it is often referred to as ‘Sanger sequencing’.

Because of the nucleotides that are used, it is also referred to as ‘di-

deoxy sequencing’.

This technique is basically just a modified DNA polymerase

reaction (like PCR), so we would perform an in vitro polymerase

reaction, with added di-deoxy nucleotides.

See the next several slides.


No 3’ --OH


If we include an

appropriate amount of

di-deoxy nucleotides

along with regular

deoxynucleotides, when

DNA polymerase is

‘reading’ a template

DNA, sometimes a di-

deoxy nucleotide will be

incorporated into the

growing strand, but most

often a regular

nucleotide will be

incorporated so the

polymerase keeps going.

If you have enough

template in a reaction

tube, you’ll end up with

polymerase products that

stop at each and every

place on the template.

Figure Q10-10 Essential Cell Biology (© Garland Science 2010)

If each of the lanes represents a single reaction tube containing a

template DNA + DNA polymerase + appropriate buffers + a

primer (remember, DNA polymerase always needs a primer to

start—in this case, we use only a single primer because we only

want to ‘read’ a single strand of the DNA [because we can deduce

the sequence of the other strand) + regular dNTPs (all 4 of them,

at least one of which is radioactively labeled) + 1 di-deoxy

nucleotide, this is what you get when you subject the products of

Sanger sequencing to gel electrophoresis. In the image of a gel to

the left, lane 1 is a reaction with di-deoxy A included; lane 2 with

di-deoxy G included; lane 3 with di-deoxy C and lane 4 with di-

deoxy T.

The gel is read from the bottom up (because the shorter fragments

go further in the gel during electrophoresis, thus those at the

bottom are closest to the primer you put in the tube).

Thus the sequence of this product is

5’-CGCGGGTCAAGTGGTTGACCT….

Test yourself to read the next 10 nucleotides.

Remember that what you are reading is the product of

polymerization—the template is complementary to this product.


More modern techniques help us avoid using radioactive nucleotides, and also help us

automate the process. Instead of radioactively-labeling nucleotides, we label them with

color; each regular nucleotide is tagged with a different color. As a polymerase product

comes off the bottom of the gel, a spectrophotometer can detect what color it was and

records that color (and thus the sequence) in a computer. You can come look at the

computer print out several hours later, and you have your sequence!

Expression cloning

This is the same thing as molecular cloning, except that the plasmid that you

will use is a bit different. It includes a promoter so that the fragment of DNA

you ligate into the plasmid can be transcribed and translated into protein.

Of course to do this, your recombinant DNA plasmid must be introduced into a

cell—either prokaryotic or eukaryotic (different introduction techniques can/are

used for different cell types).

The most difficult aspect of expression cloning is that the fragment of DNA you

ligate must be in the correct reading frame to produce your protein of interest.

See the next slide.

DNA Microarrays

DNA microarrays are used to assess the relative

expression of thousands of genes

simultaneously—relative expression means that

two things are being compared relative to one

another.

One isolates mRNA from two different sources

(the two to be compared—e.g., normal cells

and cancer cells, or lung cells of a smoker

versus lung cells of a non-smoker). The

mRNAs are converted to cDNAs using reverse

transcriptase, and the cDNAs are tagged with

different color labels, usually red fluorescent

tags on the mRNA from one source and green

fluorescent tags on the mRNA from the other.

The color-tagged mRNAs are mixed, and

incubated with a slide to which thousands of

DNA fragments are bound in a grid. Each of

the fragments is from a known gene, and there

is a ‘key’ that lets researchers know where each

of the known genes is located on the slide.


The figure to the left is from

your text book—it shows

the first four steps that are

shown in the figure on the

previous slide (from another

text book).

After binding (hybridizing)

the labeled cDNA to the

slide and washing,

theoretically the cDNAs will

bind to their complementary

DNA fragments on the slide

and stick there.

Because they are colored,

we can detect where the

cDNAs are bound.

The figure on the left is a ‘cartoon’ of the grid on the slide—each dot on the grid represents

a place where known DNA has been ‘dotted’. Again, presumably any labeled cDNA that is

complementary to one of those dots of DNA will bind there. The figure on the right shows

a grid to which the red and green-labeled cDNAs have been bound. A computer would

read this and be able to tell whether there was more red bound to a dot, more green bound

to a dot, or equal amounts of red and green (detected here as yellow). Thus, if there was

more ‘red’ cDNA, this meant that the source that gave rise to that cDNA was expressing

that gene (the one in the dot) more than the ‘green’ source; alternatively, if a dot is more

green than red, it means that the source of the green cDNA was expressing that DNA at a

higher level than the red source. If they are expressed in both sources equally, the dot will

be yellow.


The analogous figure

from your text book—this

represents the last two

steps in the figure shown

on the first ‘DNA

microarray’ slide.

Generating

Transgenic

Animals

We might do this to produce a number of different transgenic organisms (shown on the

next slide—know them!). In either case, one typical use is to determine what effect a gene

has on a particular process (so you take it out and see what happens, or you over-produce

it and see what happens).

A similar process is done with plants to produce genetically modified foods. In that case,

a foreign gene might be introduced to have the plant produce some pesticide, or to be

resistant to some herbicide.

In the next slide, the red gene represents a gene that is modified from the normal, whereas

the green gene is normal. Thus, panel c is showing something that might be used to

determine whether expression of the mutant gene might ‘over-ride’ the normal gene in a

‘dominant-negative’ manner (this is analogous to a dominant mutation in an organism—

you may have learned this in genetics class.

Generating Transgenic Animals

This is obviously a very complex process, and requires lots of steps , and hope that you can derive the

nuances of this process from reading your text book. I’m going to simply put the steps here to produce an

animal (mouse) that does not make your protein of interest. This is called a ‘knock out’ animal, and Dr.

Mario Cappechi from the University of Utah receive a Nobel Prize a few years ago for developing this

procedure.

1. Clone your gene of interest into an expression plasmid. The whole gene with introns.

2. Mutate your gene such that the transcription start site (and maybe another 1-2 exons) is deleted.

This mutated gene is still in the expression plasmid. Most people also add a ‘selectable marker’ into

the plasmid so they can tell whether or not the plasmid is present. An example of the ‘selectable

marker’ is a gene for antibiotic resistance.

3. Introduce the mutated/truncated gene into an embryonic stem (ES) cell from a brown mouse. If the

ES cells are cultured in the presence of antibiotic, then only the ES cells that contain the plasmid

will be able to grow.

4. You pick up these cells and make sure that they have your mutated gene in them. These are the steps

shown in panel A on the next slide.

5. After ensuring that you have stem cells with your mutant gene, you need to get an actual mouse

embryo. You do this by harvesting them from a white mouse. Because it’s an early embryo, we can

add in extra cells and the embryo will develop normally—this is because none of the cells in the

embryo yet ‘know’ what they’re meant to be.

6. The embryo of mixed white and brown mouse cells is now implanted into a ‘pseudopregant’ female

mouse. This just means that she’s ramped up on hormones and will ‘accept’ the implanted embryo.

This is mouse IVF.

7. The mouse delivers some baby mice. If you’re lucky, the mice will have some of their ‘parts’ that

carry the modified/mutant gene and therefore don’t’ express your gene of interest.


But wait! Step 7 above says that we hope that the

mouse babies will have some of their ‘parts’ with

the mutant gene. Actually, the only ‘parts’ we care

about are the gonads. That is because the cells in

these mouse babies have 2 copies of your gene of

interest, one that you hope is mutated (i.e.,

missing it’s transcription start site) and therefore

not being expressed. If the gonads have this

mutation, then when gametes (sex cells) are made,

the two copies of the gene (one mutated and one

normal) will be separated; some of the haploid

gametes will have the mutant gene only.

So, step 8 is to let these mouse babies grow up and

then you mate them with other normal mice. If

their gonads carried the mutation, then some of the

babies resulting from that mating will be

heterozygous for the mutant gene. They’ll still

have the normal gene, but they will be entirely

heterozygous, and carriers of the

mutation/deletion.

9. Genotype the mouse babies to see who is

heterozygous for the mutation/deletion.

10. Mate the heterozygous animals to (hopefully)

produce mice homozygous for the deletion

(assuming that the total deletion of the gene is not

lethal). And yes, you’re mating siblings! It’s gross,

but they’re mice!

That’s it. What do you think? Pretty

cool stuff, huh? I hope it all makes

sense. Again, we’ll revisit anything

you are unclear about when I return.

Till then, I hope your test went well,

don’t forget your writing

assignment/book report, and have a

great week! See you November 5th.

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