Lecture 24:Genetic Engineering:

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Dec 11, 2012 (4 years and 8 months ago)

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Lecture 24:Genetic Engineering:

PCR, RFLP Analysis & Gene Therapy

The Polymerase Chain Reaction (PCR) Can Make Millions of Copies of DNA in a
Short Time



The polymerase chain reaction (PCR) is a rapid way of amplifying (duplicating)
specific DNA sequences



Method was devised by Kary Mullis of Cetus Corporation, Emeryville

o

He recieved a $20,000 bonus and later a Nobel Prize

o

Later the patent was sold to Hoffman
-
LaRoche for $300,000,000



DNA heated to high temperature is not destroyed; separates into single s
trands,
but reforms helix when cooled



PCR Method:

o

DNA to be amplified is put into solution containing:



Short DNA "primers" which can bind to the 3' ends of the DNA



The 4 nucleotide bases: A, C, G, T



DNA polymerase



Special DNA polymerase (Taq polymera
se) working at
very high temperature is used; isolated from algae living in
hot springs

o

DNA is heated to 95 deg C
-
> single chains

o

Solution is then cooled to 55 deg C; at 55 deg primers bind to ends of the
single strand DNA

o

The temperature is now raised

to 72 deg and the DNA polymerase causes
the synthesis of new complementary strands to all the single strands

o

This is the end of the first cycle

o

The temperature is cycled from 95 to 55 to 72 over and over

o

The DNA is doubled at each cycle and at the end
of 32 cycles it has been
amplified 1 billion times

o

A cycle can be done in as little as 17 seconds, so it is possible to get a
billion
-
fold amplification in an hour or less including set
-
up time




The figures shows 2 cycles of PCR



PCR can be used to dupicate single DNA molecules



It has been used to amplify DNA samples from extinct species

Cloned DNA in Colonies Can be Identified by Trea
ting a Replica with
Complementary DNA Probes



Often it is desirable to find bacterial colonies that have specific cloned DNA
fragments, a specific gene for example



If some of the DNA sequence is known a short single strand radioactive probe can
be made to
search for the desired sequence

o

Most DNA probes have 15
-
50 bases



A mirror
-
image replica of the agar plate with the bacterial colonies is made

o

A piece of nitrocellulose paper is placed over the agar plate and pressed
down

o

Some of the bacteria are transf
erred to the nitrocellulose



The replica is treated with alkali to break up the cells and is exposed to the probe



The probe binds to DNA having a complementary sequence, labelling those
colonies that have it



Once the colonies having the desired gene have

been identified the investigator
can go back to the original plate and get living samples to culture



If a Gene is Expressed Antibod
ies Can be Used to Identify Proteins



Under certain conditions bacteria can express the protein coded by a cloned gene

o

Bacteria cannot express
genomic DNA

because it has introns

o

Can express
complementary DNA
, because it is made from messenger
RNA (introns removed)

o

Genomic DNA can be expressed in eukaryoti
c cells, such as yeast,
because they know how to handle introns



If a protein has been isolated antibodies can be made



The antibodies can be used to probe a replica plate to find colonies making the
protein



This allows the gene for the protein to be isol
ated

Blotting Techniques Help to Analyze Restriction Fragments



Electrophoresis gels can also be analyzed with DNA probes by a Southern blot



After gel is run a replica is made by blotting the DNA onto a nitrocellulose or
nylon filter



The replica is then
treated with probes that reveal specific bands



Example of a Southern blot:

o

Suppose you are interested in a gene which has 3 restriction sites for a
certain enzyme (marked by X in the picture, sample A); one of the sites
occassionally mutates and disappea
rs (sample B, from another person)

o

You have a probe which binds at the spot shown



o

You extract the DNA from your cells and cut it w
ith the restriction
enzyme

o

This produces hundreds of restriction fragments because the enzyme cuts
sites in other genes as well as the one you are interested in

o

If you do a Southern blot and then stain the DNA with a general stain such
as ethidium bromid
e you will see a smear like this because the hundreds of
bands run together



o

If you treat the replica with the probe only the bands
that bind the probe
will light up



o

The banding shows that when the mutation occurs the 15.2 kilobase band
(sample A) disappears and
is replaced by a 20.6 kilobase band (sample B)

o

Why doesn't the 5.4 kilobase band show in sample A? Look at the diagram
above to see where the probe binds.



The Southern blot is named for the man who developed the technique



Other types of blots have been
developed:

o

Northern blot: probes RNA

o

Western blot: probes protein, using antibodies

RFLPs: Different Individuals May Have Different Sizes of Restriction Fragments



RFLP = restriction fragment length polymorphism



A fancy way of saying, when you cut the g
enes up (with restriction enzymes)
different people have different sized chunks

o

The A & B samples that we discussed Southern blotting are examples: the
same enzyme gives a 20.6 kb fragment in one case, and a 15.2 plus a 5.4
kb fragment in the other case



To illustrate RFLPs further we will discuss 2 real cases, both associated with the
sickle cell anemia mutation



Case 1: HpaI sites flanking the globin gene

o

If you take the DNA from 2 different individuals and compare the
sequences in regions that do not co
de for genes you will find differences
in 0.2 to 1% of the bases

o

Most of these differences are neutral since they do not affect coding for
genes; they tend to accumulate because they are not eliminated by natural
selection

o

Sometimes these variations in b
ases can be used as markers for genes



The variation must affect a restriction site



It must be very close to a gene

o

In 1978 Kan & Dozy (UC San Francisco) reported that restriction sites of
the enzyme HpaI were associated with sickle cell anemia; they hop
ed the
polymorphism could be used for diagnosis

o

The HpaI sites are in non
-
coding regions flanking the globin gene



o

It was found that
the HpaI site downstream from the gene was different in
people with normal hemoglobin A and those with hemoglobin S



American blacks (about 60%) with hemoglobin S had a 13 kb
restriction fragment



Those with normal hemoglobin A had a 7.6 kb restriction fra
gment

o

Probable origin of the association between the 2 genes:



Step 1: a mutation occurs producing a 13 kb restriction fragment in
people with normal hemoglobin (probably in the Upper Volta
region of Africa)



Step 2: the sickle mutation occurs in a person

with the 13 kb
restriction fragment and spreads throughout West Africa &
Mediteranean

o

In other parts of the world the sickle mutation apparently occured in a
person or several persons with the 7.6 kb restriction fragment (India, Saudi
Arabia, East Africa
)

o

This RFLP is of limited value for diagnosis because in some parts of the
world sickle cell anemia is associated with the 13 kb fragment, while in
others it is associated with the 7.6 kb fragment



Case 2: MstII site in the region with the sickle point mut
ation

o

The codon region near the sickle mutation is cut by several restriction
enzymes

o

The most useful of these enzymes is MstII, which cuts in 3 places near the
mutation site:



o

The sickle mutation destroys the middle MstII site



N in the MstII site code stands for any nucleotide (it can be G, for
instance)



Hemoglobin A has an MstII site in codons 5 to 7, but changing an
A to a T destr
oys the site in hemoglobin S



o

In normal hemoglobin MstII will produce a fragment of 1.15 kb in this
region

o

When the site is mutated

the fragment enlarges to 1.35 kb

o

If you do a Southern blot test, this is the pattern you will see for people
with the different types of hemoglobin:



o

The AS individual has one gene of each type and this gives him 2 bands

o

Since the restriction site and mutation site overlap this test will give the
correct diagnosis 100% of the time (excluding experimental error)

Gene Therapy Tries t
o Replace Damaged Genes



Cloning techniqes could be used to replace damaged genes with good ones



Genes have already been inserted into animal eggs to make transgenic study
animals

o

Sickle cell anemia gene has been put into mice, for example



Gene insertion

into the germline (eggs):

o

Can be used to prevent appearance of a disease

o

Fertilized egg removed from animal

o

DNA inserted into nucleus with micropipette

o

Egg put back into female for development



o

Technical problems



Gene must insert into egg DNA; insertion is random and often fails



Gene needs control elements such as promoters
-

these may be
missing in random insertion



Attempts to t
arget genes

o

Ethical problems



Many object to directly controlling heredity in this way



Gene inserted into adult animal

o

This is is an attempt to cure a disease that is already present

o

Usually gene is put into a vector, such as a harmless virus, that can

carry
the gene into cells

o

Very difficult because millions of cells must receive the gene

For More Information

Michael King of the Terre Haute Center for Medical Education has a biochemistry course
with an excellent section on
Molecular Tools of Medicine
. Another online treatment of
DNA technology is the MIT
Biology Hypertextbook
. The Cold Spring Harbor Marine
Biology L
ab has a
DNA Learning Center

with animations of

PCR

and
Southern Blotting.

The use of restriction f
ragments in criminal investigation is illustrated in a
DNA
Detective
section.

A good concise written account of DNA technology is:

James Watson, Michael Gilman, Jan Witkowski & Mark Zoller.

Recombinant DNA
, 2nd
edition. NY: WH Freeman, 1992.

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