BIOC15 CLASS 20 JULY 13, 2017

Last class we talked about making Genomic libraries and cDNA libraries. Now lets compare the two
with the help of textbook Figure 14.9(both Can eds)/ 9.9 (US)

Genomic DNA libraries

As I already mentioned, the individual DNA clones do not usually represent functional units of
the DNA. Each clone is generated based on restriction site location.
A complete genomic library will represent all of the sequences of the genome. Since the vast
majority of the cells of the organism all have the same genomic DNA, you could make a
complete genomic library from almost any cell type of the organism.
The complete genomic library will contain DNA clones with all parts of the genes promoter,
introns, exons, and 3 and 5 untranslated regions, but all of these parts of the gene might not be
in one clone.

cDNA LIBRARIES
The individual clones DO represent functional units- the coding region of a mRNA.
Not all genes will be represented in a particular cDNA library. The cDNA library will only
contain the genes that were transcribed into mRNA in the cell type used to make the cDNA
library. Thus cDNA libraries from different cell types will not contain all of the same genes.
cDNA libraries will NOT contain promoter regions, and introns.
The more actively transcribed a gene is, the more copies of the mRNA will be present in the
cell, and the more clones within the library will exist. Figure 14.9(both Can eds)/ 9.9 (US)
lower portion.

cDNA libraries are often made when you want to create a cellular clone carrying a particular gene for
the purpose of making many copies of that gene and the genes product (a protein). You start by
making a cDNA library for a tissue of the body that you know expresses the gene. The library is the
start, but after that you have to screen the library for the one member of the library that contains the
gene of interest.

GENOMIC VERSUS cDNA LIBRARIES.

Figure 14.9(both Can eds)/ 9.9 (US)
Genomic libraries were the first kind made and were used to try to clone a particular gene. Nowadays
genomic libraries are not needed to find and clone genes (since cDNA libraries are easier to manage),
UNLESS you want to look at the promoter or introns. Promoters and introns are not in the cDNA
library since the starting material is mature mRNAs.

The big usage of genomic libraries is for the sequencing of entire genomes. We will come back to this
topic in a future lecture.

NOW LETS LOOK AT HOW YOU CAN SCREEN A LIBRARY TO FIND A PARTICULAR
CLONE. WE WILL LOOK ONLY AT THE TWO SIMPLEST TECHNIQUES FOR SCREENING A
LIBRARY.

SCREENING AN EXPRESSION LIBRARY WITH AN ANTIBODY SPECIFIC FOR THE GENES

PROTEIN PRODUCT.
One way to screen an expression library is to use an antibody as a probe.

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How is this accomplished? The cloned sequence is DNA, and antibodies work by identifying proteins;
how can you use the antibody to find a DNA clone?

The process can be done if the cloned sequence was a cDNA put into an expression vector. This means
that the cloning site of the vector is designed to put the cDNA right next to a promoter that works well
in the host cell.

By using an expression vector for the cloning, then the bacterial host cell will be able to make the
cloned genes protein. What else do you need?

To screen an expression library for the cDNA of interest, you also need an antibody that recognizes and
binds tightly to the cDNAs protein product.

How would you get an antibody? If the protein was purified, then you can inject the purified protein
into a rabbit or rat. If you are lucky, the rabbit/rat may respond to the injection by creating antibodies
specifically designed to recognize the injected protein.

These antibodies can be purified from the rat/rabbits blood. Once you have the antibody you can use
it to screen an expression library. How?

Lets look at Figure 9.12(old US edition), powerpoint slide #28).

The screening starts with colonies on a plate. You make a replica of the plate for archiving.

A filter is laid over the original plate transferring some of the cells from each bacterial colony onto the
paper. The position of the filter on the plate is carefully marked.

The cells on the filter are lysed. Once the proteins have been immobilized on the filters, the filter is
probed with an antibody specific for the protein of interest. The antibody itself can be labeled, and
detected by a variety of means, most typically a fluorescent molecular is covalently linked to the
antibody).

The antibody can be used to recognize a specific colony that contains the gene of interest. The antibody
can detect the protein produced by the gene of interest. That is why the library must be made using an
expression vector.

Thus screening a library with an antibody is a fairly simple way to screen a library but you must have
made the library using cDNA that was inserted into an expression vector and secondly you must have
an antibody directed specifically against the genes protein.

The other relatively easy way to screen a library is using DNA hybridization. This is the easiest and
most robust way to screen a library, but you need to have a complementary labeled probe for it to work.
You will want a hybrid nucleic acid to form and be detected.

SCREENING A LIBRARY WITH A LABELED NUCLEIC ACID PROBE THAT IS

COMPLEMENTARY TO THE DNA SEQUENCE OF INTEREST- i.e. USES HYBRIDIZATION.

This screening technique is illustrated in Figure 9.13 (old US edition) and on powerpoint slide for
screening a library using a homologous probe.

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1. You start with the host cells (with the recombinant DNA within it), growing as separate colonies of
cellular clones, on culture dish.

2. You make a replica of the original plate for archiving.

3. In the next step you very carefully put a piece of nitrocellulose over the colony, some of the cells
will be transferred to the paper.

4. Then you lyse the bacterial cells on the nitrocellulose paper and you denature the DNA, and then
immobilize the DNA onto the paper with heat or UV light. Before you lift the filter off the Petri dish,
you carefully mark it so you can know its position relative to the original plate.

5. Now you hybridize the filter with a labeled probe that has a nucleic acid sequence complementary to
the gene you are interested in.

A labeled probe is the name we give to the nucleic acid molecule that is labeled either with
radioactivity, or chemically altered DNA, that allows you to detect it.

Labeled probes can be made by doing DNA replication in a test tube, and some of the nucleotides are
tagged with a radioactive or fluorescent nucleotide.

If there was a colony on the plate that had the gene you were interested in then the DNA from that
colony will hybridize with your probe, and can be visualized.
If the probe was labeled radioactively you detect the colonies by autoradiography. For auto-
radiography a piece of X-ray film is placed over the filter. The sites on the filter that contain
radioactivity excite the silver grains on the film, and when the film is developed we can see
which colonies contain radioactivity. These are the colonies that contain recombinant DNA that
we are interested in.

6. Since you carefully marked the paper, you can go back and identify the colony that donated the DNA
that gave the hybridization, and so identify the clone that is carrying at least a portion of the gene you
want.

This is the principle of screening using a nucleic acid probe.

Whats the catch here?

The catch is that in order to screen you need to have a probe that is complementary to the sequence of
your gene. How do you get this?
There are a variety of possibilities.

a) One possibility is that someone else has already cloned a DNA sequence similar to the gene you are
interested in. e.g. A cloned Drosophila vision gene might help the researcher find a vision gene in
humans.

b) Another possibility is that another gene has been cloned that shares a functional domain with your
gene. Therefore the portion of the cloned gene that is similar can be used as a complementary probe.

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c) Yet another method, and the one that is often used, when you cant get a homologous sequence from
someone else, is to create a probe based on information about the protein the gene encodes.

If you can purify the protein, then you can determine the identity of a sequence of the amino acids in a
portion of the protein. Once you know the amino acid sequence you can use your knowledge of the
genetic code to create a synthetic DNA - an oligonucleotide with a DNA sequence that should
hybridize with the gene that was responsible for encoding the protein. Figure 14.10 (both Can eds)/
9.10 (US). The process of designing probes that will be complementary to the gene, based on the
amino acid sequence is called reverse translation. (reverse translation is NOT a naturally occurring
process, but a molecular, computer assisted technique)

In general, screening a library using a complementary probe requires some knowledge of the genes
likely DNA sequence or its protein product. Thus the first two screening techniques require either
knowledge of the DNA sequence, or knowledge of the amino acid sequence or the availability of an
antibody that can specifically detect the genes protein product.

There are other, more complex schemes that can be used sometimes to screen libraries when you have
no knowledge of the protein or gene. We wont go into these techniques AND these techniques are not
needed so often any more since so many genomes have already been sequenced. Now scientists search
for the genes of interest by hunting through the published sequence of genomic libraries rather than
hunting through a genomic library.

Once a scientist finds a candidate gene, they look for additional evidence that it is the gene they are
interested in.

We just looked at one use of DNA hybridization (to screen a genomic library), but there are many other
usages; lets look at two more important techniques using hybridization Southern blotting and
Northern blotting.

SOUTHERN BLOTTING.
Southern Blotting is a technique that combines restriction digestion, gel electrophoresis of the cut
DNA fragments and nucleic acid hybridization. Figure 14.11(Can)/9.11 (US), not in 2nd edition, see
ppt slide 61&62.

The first step in the process is to prepare the DNA samples. Often genomic DNA is cut with specific
restriction fragments. It could be done with a complete or a partial digestion, but for most application
the digestion is complete.

The DNA samples are then loaded into an agarose gel and run using gel electrophoresis.

If you just cut up genomic DNA and run the gel, and stain the gel with ethidium bromide you can not
identify specific DNA segments.

What you would see would look a lot like that seen in Figure 14.11(Can)/9.11 (US). You would see a
smear of DNA fragments in a continuous range of sizes.

One reason for doing Southern blotting is to identify where in the gel (what size), is a particular DNA
segment of interest. How can you find one particular DNA segment in the gel?
The answer is a Southern Blot. (named after its inventor, Dr. Southern).
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The next step is to take an unstained gel, in which the DNA has been run, and transfer the separated
DNA fragments from the gel to special paper such as nitrocellulose. The technique for doing this is
seen in the bottom half of Figure 14.11(Can)/9.11 (US).

The gel is put on a sponge platform. Then the nitrocellulose paper is placed precisely over the gel.
Then paper towels are put on top of the nitrocellulose.

Capillary action of water moving from the sponge to the paper towel, moves the DNA out of the porous
gel and traps it on the nonporous nitrocellulose paper.

The transfer procedure maintains the DNAs precise relative position. For example, if a particular
DNA segment was at the top of the gel, it will be transferred to the top of the paper).

Next you can treat the paper in a way that traps the DNA on it, but allows the DNA strands to be
opened up, this denatures the genomic DNA into single stranded segments.

Now we are ready to try to find the location of a particular DNA segment. It will have a characteristic
length, but how can we find it?

We need to have a complementary, labeled, nucleic acid that we call the probe.

Once we have the labeled probe, we can perform hybridization with the probe and the DNA
immobilized on the paper. If there is DNA complementary to the probe, on the paper, the probe can
find it.

Thus Southern blotting is designed to determine if a particular sequence was represent in the genome
(or partial genome) that was cut with restriction enzyme, and to know what size the piece is.

The key feature of Southern blotting is its specificity and its sensitivity. The specificity comes from the
fact that the location of the fragment of interest can be determined by the fact that it binds the labeled
probe. The increased sensitivity of the reaction comes from the fact that the probe has many copies of
the tag on it, and so amplifies the signal. You need 1000-fold less DNA in the band to be detected by
hybridization, than could be detected by ethidium bromide staining.

Southern blotting can be used in medical diagnostic testing. But we will talk about that a little later in
the course.

NORTHERN BLOTTING
Northern Blotting is a variation of Southern blotting. For Northern blots the starting material is a
population of RNA. Thus Northern blotting requires the purification of RNA, then the separation of the
RNA molecules in a gel through gel electrophoresis. Then through nucleic acid hybridization with a
labeled probe, you can identify a particular RNA within the blot. Northern blotting is a very commonly
used technique.

For Northern blotting RNA, typically mRNA is carefully prepared from specific types of cells. Since
mRNAs are usually relatively small in size, they do not need to be cut with restriction enzymes. The
purified mRNAs can simply be run on a gel to separate mRNAs of different sizes. The RNA is then
transferred to paper and a nucleic acid probe can then be used to see if the cells were making that RNA.
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Homework Problems
Hasenkampf PRACTICE QUESTIONS
Practice question 1
a) How could you screen a library if you knew the amino acid sequence of part of the protein?
b) What type of library (genomic or cDNA) would you screen?

Practice question 2
a) How could you screen a library if you had an antibody to part of the protein?
b) What type of library (genomic or cDNA or cDNA expression) would you screen?

Practice question 3
Using a cDNA library, you isolate two different cDNA clones that hybridized with your probe. The
beginning and the ending of the sequences of the two clones are the same, but the middle sequence is
different. How can you explain the different cDNAs?

Practice question 4
What sequence information is lacking in a cDNA library?

Textbook Problems
Both Canadian edition Chapter 14 : problems 1, 2, 3,4, 6, 7, 8, 10, 11b, 12, 14.
US 4th Edition Chapter 9: problems 1, 2, 3,4, 6, 7, 8, 10, 11b, 12, 14.

The Polymerase Chain Reaction

At the beginning of the section on recombinant DNA technology I indicated there were five basic tools
nucleic acid hybridization
cutting nucleic acids into manageable size pieces,
separating the pieces based on size
making many copies of a particular gene
by molecular cloning
by polymerase chain reaction
DNA sequencing
Making sense of sequencing data

Now I want to talk about PCR. PCR is another way to make many copies of a particular DNA
sequence.

PCR stands for the polymerase chain reaction.

PCR is a specialized version of in vitro DNA replication. The purpose of PCR is to preferentially
amplify a particular DNA target sequence, even when the desired sequence is part of an intact part
of the genomic DNA.

Figure 14.12 (Can)/ 9.12 US shows the principles of the technique.

The key to the PCR technique is being able to create specific DNA primers, that bracket the DNA
target sequence.

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Because DNA replication requires primers, the only thing that can be replicated is the region that is 3
of the primer, and the only dsDNA that can be amplified by PCR is a region of DNA bracketed by two
such primers. Figure 14.12 (Can)/ 9.12 US. (first page of the figure)

Specific primer pairs will direct the preferential replication of just the specific DNA sequence that is
between the sites of the primer binding. The primers are shown on the left, top side of Figure 14.12
(Can)/ 9.12 US. (first page of the figure)

One cycle of PCR is shown in the lower half of the left side of the figure. We denature the DNA, and
add our primers, and then allow DNA replication to occur.

For this DNA replication we use a very special DNA polymerase that comes from a microorganisms
that lives in hot springs (the organisms is Thermus aquaticus, and its DNA polymerase is called Taq
polymerase).

Because this organism grows at high temperatures, its DNA polymerase (Taq polymerase) is not ruined
by high heat like a normal DNA polymerase would be. This characteristic of Taq polymerase is what it
allows one to do many rounds of DNA replication. You replicate, then denature the DNA by heat, add
primers again, replicate, denature add more primers, replicate, denature etc. etc. With each round of
replication more templates are being created. (2, 4, 8, 16, 32, 64, 128, etc)

These repeated round of synthesis result in an exponential increase in the amount of the specific
sequence being created. A million-fold amplification is commonly achieved with 22 cycles of
replication. Figure 14.12 (Can)/ 9.12 US. (second page of the figure)

Thus the few cells at the base of one hair follicle can be used in PCR to get a million copies of a
specific region of the DNA - enough copies can be obtained to sequence a gene, or to make labeled
probes.

What I do not like about Figure 14.12 (Can)/ 9.12 US (page 2) is that it only shows you the one DNA
region of interest. However you should keep in mind that PCR will work to amplify the one DNA
fragment even if it is part of a larger DNA molecule or if there are other DNA molecules present
initially.

The preferential amplification of the region of interest will occur, so long as the primers can base pair
to DNA regions that bracket the region of interest.

PCR is an incredibly powerful technique, with many applications. PCR is great but remember PCR
amplification of a specific DNA segment is only possible once you know the DNA sequence at the
boundaries of the gene of interest.

Now that so many species have had their genomes sequenced, in such organisms it is usually easy to
design primers that will preferentially amplify a specific region of a chromosome.

PCR CLONING.
If you DO know the DNA sequence of the gene, or even the sequence at just the beginning and end of
the gene, you can clone it quickly using PCR as the first step.

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1) You can take a small amount of genomic DNA and with the primers create millions of copies of the
one PCR amplified sequence, purify it on a gel, and then put it into the vector you want.

2) You then take the amplified gene of interest and insert the amplified fragment into a vector.

3) Next you can transform the vector into a host cell.

4) Next you screen. In this case all you have to do for screening is select for host cells that have
incorporated a vector with recombinant DNA. You dont create or screen an entire library. You
directly create the one clone of interest.

Thus there are two types of ways to create DNA clones; one way is PCR cloning and the other is
molecular cloning. PCR cloning is by far the easier method, but the ability to do it hinges on
being able to design primers specific for the region of interest.

The Polymerase Chain Reaction

At the beginning of the section on recombinant DNA technology I indicated there were five basic tools
cutting nucleic acids into manageable size pieces,
separating the pieces based on size
nucleic acid hybridization
DNA sequencing and
making many copies of a particular gene
by molecular cloning
by polymerase chain reaction

Now I want to talk about this last technique of PCR. PCR is another way to make many copies of a
particular DNA sequence.

PCR stands for the polymerase chain reaction.

PCR is a specialized version of in vitro DNA replication. The purpose of PCR is to preferentially
amplify a particular DNA target sequence, even when the desired sequence is part of an intact part
of the genomic DNA.

Figure 14.12 (Can)/ 9.12 US shows the principles of the technique.

The key to the PCR technique is being able to create specific DNA primers, that bracket the DNA
target sequence.

Because DNA replication requires primers, the only thing that can be replicated is the region that is 3
of the primer, and the only dsDNA that can be amplified by PCR is a region of DNA bracketed by two
such primers. Figure 14.12 (Can)/ 9.12 US. (first page of the figure)

Specific primer pairs will direct the preferential replication of just the specific DNA sequence that is
between the sites of the primer binding. The primers are shown on the left, top side of Figure 14.12
(Can)/ 9.12 US. (first page of the figure)

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One cycle of PCR is shown in the lower half of the left side of the figure. We denature the DNA, and
add our primers, and then allow DNA replication to occur.

For this DNA replication we use a very special DNA polymerase that comes from a microorganisms
that lives in hot springs (the organisms is Thermus aquaticus, and its DNA polymerase is called Taq
polymerase).

Because this organism grows at high temperatures, its DNA polymerase (Taq polymerase) is not ruined
by high heat like a normal DNA polymerase would be. This characteristic of Taq polymerase is what it
allows one to do many rounds of DNA replication. You replicate, then denature the DNA by heat, add
primers again, replicate, denature add more primers, replicate, denature etc. etc. With each round of
replication more templates are being created. (2, 4, 8, 16, 32, 64, 128, etc)

These repeated round of synthesis result in an exponential increase in the amount of the specific
sequence being created. A million-fold amplification is commonly achieved with 22 cycles of
replication. Figure 14.12 (Can)/ 9.12 US. (second page of the figure)

Thus the few cells at the base of one hair follicle can be used in PCR to get a million copies of a
specific region of the DNA - enough copies can be obtained to sequence a gene, or to make labeled
probes.

What I do not like about Figure 14.12 (Can)/ 9.12 US (page 2) is that it only shows you the one DNA
region of interest. However you should keep in mind that PCR will work to amplify the one DNA
fragment even if it is part of a larger DNA molecule or if there are other DNA molecules present
initially.

The preferential amplification of the region of interest will occur, so long as the primers can base pair
to DNA regions that bracket the region of interest.

PCR is an incredibly powerful technique, with many applications. PCR is great but remember PCR
amplification of a specific DNA segment is only possible once you know the DNA sequence at the
boundaries of the gene of interest.

When it was invented PCR was limited because in many cases the DNA sequence bracketing the gene
of interest was not known, and so primers could not be designed. Now that so many species have had
their genomes sequenced, in such organisms it is usually easy to design primers that will preferentially
amplify a specific region of a chromosome. SO PCR is an every day technique now.