iochemical and Molecular Methods

m Hybridization techniques allow identification of

specific macromolecules, e.g. proteins, and
DNA or RNA sequences based on ability of NA
to bind specifically to labeled, known, single-
stranded NA sequences.
m Separation methods employed range from
enzyme digestion, chromatography,
electrophoresis or centrifugation.
m Using restriction endonucleases which cleave
DNA at specific sequences, recombinant DNA
and other facets of biotechnology are benefited
by these methods.
m À

 
  

 Nucleic Acid Hybridization. (A) If the DNA helix is separated into 2 strands,
the strands should reanneal, given the appropriate ionic conditions and
time. (B) If DNA is separated into its 2 strands, RNA should be able to bind to
the genes that encode it. If present in sufficiently large amounts compared
with the DNA, the RNA will replace one of the DNA strands in this region.
ombined with
reporter
molecules,
hybridization
enables
cytogeneticists
to create
probes to
detect ,
quantify,
visualize
DNA/RNA
location, and
monitor their
activity in a
given cell/
tissue of
interest.
©he synthesis of cDNA
A DNA copy (cDNA) of
an mRNA molecule is
produced by the
enzyme reverse
transcriptase, thereby
forming a DNA/RNA
hybrid helix. © eating
the DNA/RNA hyb id
with alkali o
ising the
pH of the solution
den
tu es the double-
st
nded hyb id
nd
cle
ing the RNA. ©he
em
ining single-
st
nded cDNA is then
copied into double-
st
nded cDNA by the
enzyme DNA
polyme
se.
 A cDNA library can
be constructed using a
bacteriophage vector.

¦lating of the recombinant phage

on a lawn of E. coli generates a
set of cDNA clones representing
all the cellular mRNAs.
 ¦hage cDNA libraries can
be screened with a
radiolabeled probe to identify a
clone of interest.
©he appearance of a spot on the
autoradiogram indicates the
presence of a recombinant clone
containing DNA complementary to
the probe. ©he position of the spot
on the autoradiogram is the
mirror image of the position on the
original petri dish of that
particular clone. Aligning the
autoradiogram with the original
petri dish will locate the
corresponding clone from which
infectious phage particles can be
recovered and replated at low
density, resulting in well-separated
plaques. ¦ure isolates eventually
are obtained by repeating the
hybridization assay.
 ¦rotocol
used to make
cDNA
libraries:
¦R
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 ©he use of ¦R in
forensic science
(A) A ¦R re
ction using
2 primers th
 br
cke

p
ricul
r micros
ellie,
or VN©R sequence,
produces
differen p
ir
of DNA b
nds from e
ch
indiidu
. E
ch b
nd
represens VN©R
sequences inheried
from he moher & f
her.
(B) DNA b
nds ob
ined
from
se of differen
¦R re
cions, e
ch of
which
pifies he DNA
fro
differen VN©R
sequence, c
n sere
s

"fingerprin" o idenify
e
ch indiidu
 ne
r
unique. ©he s
ring

eri
 for he ¦R
re
cion c
n be
singe
h
ir h
 w
s ef
 he
scene of
crie.
 ©he differences between cDNA clones and genomic DNA clones
Gene A is infrequently transcribed while gene  is frequently transcribed, and
both genes contain introns (green). In the genomic DNA clones both the
introns and the nontranscribed DNA are included, and most clones will
contain only part of the coding sequence of a gene. In cDNA clones, the
intron sequences have been removed by RNA splicing during the formation of
the mRNA, and a continuous coding sequence is therefore present.
 Methods for mRNA
m Isolate populations of mRNA that
characterize certain cell types and are
absent in all others
m Determine the temporal and spatial locations
of RNA expression
1.Northern blot- extract total mRNA from the
specimen and separate it by electrophoresis.
m ands produced are complementary mRNA
to the probe, and intensity of bands are
proportional to amount of specific mRNA.
Northern blotting.
 .Ribonuclease protection
3¦labeled probe is hybridized in
solution with total RNA from specimen
Hybrids are digested with ribonucleases
(double-stranded ones will be protected
from digestion)
©he mixture is separated
on a sequencing gel
Visualized radioactivity reveals a
range of intensity proportional to the
content of specific RNA in the sample.
 3.Reverse-transcription polymerase chain
reaction (R©-¦R)
©otal RNA from sample is reverse-transcribed
into cDNA using reverse transcriptase.

©wo oligonucleotides are added, chosen to

correspond to sequences in the target cDNA.

©he sequence between the primers is amplified by

repeated cycles of synthesis, melting and hybridization.

©he reaction mixture is run on a gel and the

DNA bands are visualized in the usual way.

©he intensity of bands bears some relation to

the initial amount of mRNA in the sample.
 Methods for DNA
1.In situ hybridization- designed to reveal the
spatial domains of gene expression in a
specimen.
m An antisense probe is synthesized in vitro
complementary to the mRNA to be detected.
©his is hybridized to the specimen and then
visualized.
m ¦robes usually include extra chemical groups
recognizable by a commercially available
antibody for detection (e.g. digoxigenein or DIG,
a plant sterol)
m Radioactive probes are used in radioactive in
situ hybridization (RISH), fluorescent dyes are
used in FISH.
Fluorescence In Situ
Hybridization (FISH)
begins with a DNA
probe and a target
sequence. The DNA
probe is labeled by
indirect labeling
(left) and direct
labeling (right). The
labeled probe and
the target DNA are
denatured to yield
single stranded
DNA. They are then
cobined, which
allows the annealing
of copleentary
DNA sequences.
 xn situ hybridization performed on a whole chick
embryo that have been fixed without being
sectioned. ©he probe used recognizes the mRNA
encoding Pax6 in the chick embryo. ©his probe is
labeled not with a radioactive isotope, but with a
modified U©P. ©o create this probe, a region of the
cloned Pax6 gene was transcribed into mRNA,
containing U©P conjugated with digoxigenin. ©his
does not interfere with the coding properties of
the resulting mRNA, but does make it recognizably
different from any other RNA in the cell.
.Southern blot- to evaluate DNA extracts
from tissue samples.
m ©his is a type of nucleic acid hybridization
test in which single-stranded DNA from two
sources interact.
m Strands with similar nucleic acid sequences
will anneal by base pairing (A with ©, and G
with  to form double-stranded molecules.
m One of the single-stranded DNA molecules
is a unique portion of the gene of interest,
and is radioactively labeled so it can be
detected on photographic film (the probe.
 Southern blotting. (1 DNA is treated with restriction
enzymes, and the resulting restriction fragments of DNA are
placed in a gel. ( After the fragments of DNA are separated
on the gel by electrophoresis, the DNA is denatured into
single strands. (3 ©he gel is then placed on a support on top
of a filter paper saturated with high-ionic-strength buffer.
Nitrocellulose paper or a nylon filter is placed on the gel, and
towels are placed atop the filter. ©he transfer buffer makes
its way through the gel, nitrocellulose paper, and towels by
capillary action, taking the DNA with it. ©he single-stranded
DNA is stopped by the nitrocellulose paper. (4 lot is
incubated with radioactive or fluorescent probes in sealed
bag. (5 ©he positions of the DNA in the paper directly reflect
the positions of the DNA fragments in the gel.
 B e d ess of Whie Ho se ep
ee M
c
Ws

s
ed w see
 
e US P ese B C

 Southern blots (zoo blots of various organisms. DNA using a
raioactive probe from the Antennapeia gene of Drosophila melanogaster.
Autoraiograph shows that Drosophila genes contain several portions that
are like Antennapeia genes in structure an that man organisms contain
several genes that will hbriize this raioactive gene fragment, suggesting
that Antennapeia-like genes exist in these organisms. The numbers besie
the blots inicate size of bans, in kilobases.
 3.MxROARRAYS provide a means to measure &
monitor the expression of thousands of genes at once

...\..\..\genetics\videos\dnaarray.exe
 ME©HODS OF REOMxNAN© DNA
©HNOLOGY
Knowledge of the molecular biology of cells
makes it possible to experimentally move from
gene to protein and from protein to gene!
1. acterial plasmids are small, circular, self-
replicating, extra-chromosomal DNA pieces that can
be altered in vitro by inserting or deleting specific
sequences, using restriction endonucleases.
m ecause they can be used to create clones of genes,
plasmids are called LONxNG V©ORS.

...\..\..\genetics\videos\restriction.exe
©ransfection-
cells are
incubated in
solution that
makes them
´drinkµ in
cloned DNA
usually mixed
with antibiotic
resistance
genes
lectropo
lation-
high-
voltage
pulse
´pushesµ
the
cloned
DNA into
cell
m Microinjection- cloned gene in
solution is injected into the cell
nucleus. ©he DNA is injected into
the fertilized ovum before the
male and female pronuclei have
fused, increasing the probability
that all of the cells of the
organism will harbor the gene.
m A ´gene gunµ is also used which
fires plastic bullets filled with
DNA-coated metallic pellets.
Some may penetrate the nuclei
of cells, where the introduced
DNA integrates into the DNA of
the recipient·s genome.
m ©ransposable element or retroviral vector-
cloned DNA is inserted into mobile regions of
DNA that has the ability to integrate
themselves into the genome of an organism.
m Known as
jumping
genes since
they can
move about
on the
chromosome
or among
chromosomes.
 ©ransgenic mice are
produced by random
integration of a foreign gene
into the mouse germ line.
Foreign DNA injected into
one of the two pronuclei (the
male and female haploid
nuclei contributed by the
parents has a good chance
of being randomly integrated
into the chromosomes of the
diploid zygote. ecause a
transgene is integrated into
the recipient genome by non-
homologous recombination,
it does not disrupt
endogenous genes.
m After birth, tissue samples of the young are assessed for
the presence of the desired gene. DNA from germ line
cells is given special attention.
m xf the novel gene is present in these cells, the animal and
its stem cells can be used as a founder for breeding.
m Such animal models allow researchers to test
therapeutic compounds and study the molecular basis of
given diseases.
m Mouse disease models now exist for cystic fibrosis, beta-
thalassemia, atherosclerosis, retinoblastoma, and
Duchenne muscular dystrophy.
m Somatic cells may also be grown in cell culture and
genetically modified by fusion with the enucleated egg.
m With donor DNA for cloning derived from cultured
recombinant cells, it becomes possible to carry out
specific genetic modifications and introduce the
modified genes into animals and plants.
.HxMRAS- isolating embryonic stem (S cells and
incorporating them into recipient embryos resulting into
cells of different genetic constitution appearing in the
same organism. S cells treated as transgenics have
been useful in determining how genes are regulated
during development of mice.
m ©otipotent xM blastomeres or embryonic
stem cells (S cells are cultured in vitro,
treated, injected, & integrate into another
early-stage mouse embryo. ©he result is a
chimeric mouse.
m xf the treated cells become part of the germ
line of the mouse, some of its gametes will
be derived from the donor cell.
m An isolated gene can be inserted into a
retroviral vector which infect mouse cells.
m xf such a chimeric mouse is mated with a
wild-type mouse, some of its progeny will
carry one copy of the inserted gene.
m When these heterozygous progeny are
mated to one another, about 5% of the
resulting offspring will carry copies of the
inserted gene in every cell of their bodies
 ©oday,
transgenics are
becoming such a
common
commodity.
GMO technology
comes up with
new wonders
every now and
then, it is not
surprising to have
one of these in
the future!
3. GN ©ARG©xNG (KNOK-OU© ¦RxMN©S-
wild type alleles are replaced with mutant ones.
©here are  types of mutations used in these
experiments:
a. Loss of function mutation- protein product of the
mutant gene is less active than the wild type
b. Gain of function mutation- mutant gene interferes
with the function of the wild-type form (mutant
can cause receptor activation in the absence of a
ligand-receptor complex, or a mutant
transcription factor may be active all the time and
not respond to
regulation
m ©o analyze the role of M¦ in
development, a bacterial gene for
neomycin resistance is inserted into
M¦ , destroying its ability to
function.
m ©he mutant M¦ genes are inserted
into neomycin sensitive S cells,
heterozygous S cells are then
microinjected into mouse blastocysts,
resulting to chimeras which are then
mated to wild-type mice.
m Heterozygous mice are inbred,
producing mutant mice which lacked
eyes and kidneys.
xn the absence of the M¦ protein, cells that
form the eyes & kidneys stop dividing and die.

Gene targeting makes transgenic mice that are

missing specific genes. xn this way gene targeting
can be used to analyze the roles of particular
genes during mammalian development.
...\..\..\genetics\videos\gene targeting.exe
4. AN©x- SNS RNA-
interrupts translation
of mRNA to protein by
introducing single
strands of RNA
targeted to bind with
the mRNA
m Generated by cloning
DNA into vectors with
promoters at both ends
of the inserted gene.
m When incubated with
RNA polymerase and N©¦s, the promoter will transcribe the
message in the wrong direction.
m ©ransgenic tomatoes have been constructed that carry in
their genome an artificial gene that is transcribed into an
antisense RNA complementary to the mRNA for an enzyme
involved in ethylene production. ©hese tomatoes make only
10% of the normal amount of the enzyme.
 ©he double-stranded
RNA molecules are
recognized by an RNase
and degraded into short
fragments.

©his antisense RNA will

bind to a normal cellular
message. mRNA
produced by this gene
will also be degraded.

RNA-dependent RNA
polymerase can amplify
these fragments, which
can be transmitted to
progeny cells.
Results are similar to
knock-out experiments
where the expression of
a cellular gene is
experimentally shut off.
 DNA microarray analysis
can reveal differences in
gene expression in yeast
cells under different
experimental conditions.
cDNA prepared from mRNA
isolated from wild-type
Sacc aromyces cells grown on
glucose or et anol is labeled
wit different fluorescent dyes.
xf a spot is , expression
of t at gene is t e same in
cells grown eit er on glucose
or et anol. xf a spot is ,
expression of t at gene is
greater in cells grown in
glucose. xf a spot is ,
expression of t at gene is
greater in cells grown in
et anol.
http://www.dnalc.org/resources/
animations/cloning101.html

http://www.dnalc.org/resources/
animations/gelelectrophoresis.
html

http://www.dnalc.org/resources/
animations/pcr.html

http://learn.genetics.utah.edu/c
ontent/labs/extraction/

http://learn.genetics.utah.edu/c
ontent/labs/microarray/

http://www.dnalc.org/resources/
animations/dnaarray.html