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Manipulatingnucleicacids 111109075650-phpapp02
1.
2. Biochemical and Molecular Methods
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.
Separation methods employed range from
enzyme digestion, chromatography,
electrophoresis or centrifugation.
Using restriction endonucleases which cleave
DNA at specific sequences, recombinant DNA
and other facets of biotechnology are benefited
by these methods.
Wait until next semester for this…
3. 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.
4. Combined 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.
5. Construction of a human
genomic DNA library. A
genomic library is usually
stored as a set of bacteria,
each bacterium carrying a
different fragment of human
DNA. The entire collection of
clones derived from one mRNA
preparation
constitutes a cDNA library.
Because the cells of different
tissues produce distinct sets
of mRNA molecules, a distinct
cDNA library is obtained for
each type of cell used to
prepare the library.
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6. The synthesis of cDNA.
Total mRNA is extracted from a
particular tissue. The enzyme
reverse transcriptase produces
DNA copies (cDNA) of the
mRNA molecules. A short
complement to the poly-A tail at
the 3' end of the mRNA acts as
a primer for the reverse
transcriptase, which then
copies the RNA into a
complementary DNA
chain, thereby forming a
DNA/RNA hybrid helix. Treating
the DNA/RNA hybrid with
RNaseH creates nicks and gaps
in the RNA. DNA polymerase
used to synthesize the 2nd DNA
strand synthesize the bound
RNA molecule, resulting to
sequences at the 5„ end to be
absent from cDNA libraries.
7. Gene A is infrequently
The differences between cDNA clones transcribed compared to
and genomic DNA clones derived from gene B. In the genomic
the same region of DNA. DNA library, both the
introns (green) and the
nontranscribed DNA
(pink) are included in the
clones. Most clones
contain only part of the
coding sequence of a
gene (red). In the cDNA
clones, the intron
sequences (yellow) have
been removed by RNA
splicing during the
formation of the mRNA
(blue). Because gene B is
transcribed more
frequently than gene A in
the cells from which the
cDNA library was made, it
is represented much more
frequently than A in the
cDNA library. In contrast,
A and B are in principle
represented equally in the
genomic DNA library.
8. Preparation of a bacteriophage λ cDNA
library. A mixture of mRNAs is isolated
and used to produce cDNAs
corresponding to all the cellular mRNAs
(1–3). These single-stranded cDNAs (light
green) are then converted into double-
stranded cDNAs, which are treated
with EcoRI methylase to prevent
subsequent digestion by EcoRI (4 –6 ). The
protected double-stranded cDNAs are
ligated to a synthetic double-
stranded EcoRI-site linker at both ends
and then cleaved with the corresponding
restriction enzyme, yielding cDNAs with
sticky ends (red letters); these are
incorporated into λ phage cloning
vectors, and the resulting recombinant λ
virions are plated on a lawn of E. coli cells
(7–9 )
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9. Phage cDNA libraries can
be screened with a
radiolabeled probe to identify a
clone of interest.
The appearance of a spot on the
autoradiogram indicates the
presence of a recombinant clone
containing DNA complementary to
the probe. The 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. Pure isolates eventually
are obtained by repeating the
hybridization assay.
10. Use of PCR to obtain a (A) PCR primers that flank the
stretch of DNA to be cloned are
genomic or cDNA clone. added to purified chromosomal
DNA, and many PCR cycles of
are completed. Since only the
DNA between the primers is
amplified, PCR provides a way
to obtain a short stretch of
chromosomal DNA selectively
in a virtually pure form. (B) To
use PCR to obtain a cDNA
clone of a gene, mRNA is first
purified from cells. The first
primer is then added to the
population of mRNAs, and
reverse transcriptase is used
to make a complementary DNA
strand. The 2nd primer is
added, and the single-stranded
cDNA molecule is amplified
through many PCR cycles. For
both types of cloning, the
nucleotide sequence of at least
part of the region to be cloned
must be known beforehand.
11. Cloning genomic DNA by the PCR technique. Each cycle of the reaction
begins with a brief heat treatment to separate the two strands
(aka, heat cycler). Hybridization to complementary sequences in the
two DNA strands take place to produce 4 dsDNA molecules, and the 5-
min. cycle is repeated 20-30x to produce amplified DNA copies. Trace
amounts of RNA can be analyzed in the same way by first transcribing
them into DNA with reverse transcriptase.
12. ......geneticsvideos http://highered.mcgraw-
pcr.exe hill.com/olc/dl/120078/m
icro15.swf.
A PCR reaction using 2 primers that bracket a particular
microsatellite, or VNTR sequence, produces a different pair
of DNA bands from each individual. Each band represents
VNTR sequences inherited from the mother and father.
Although some individuals have several bands in common,
the overall pattern is quite distinctive for each.
13. The PCR cloning Bands obtained from a set
technique has of PCR reactions which
largely replaced amplifies different VNTR
Southern blotting sequences, can serve as
for the diagnosis a "fingerprint" to identify
of genetic each individual. The
diseases and for starting material for the
the detection of PCR reaction can be a
low levels of viral single hair or blood left at
infection. the crime scene.
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14.
15. Methods for mRNA
Isolate populations of mRNA that
characterize certain cell types and are
absent in all others
Determine the temporal and spatial locations
of RNA expression
1.Northern blot- extract total mRNA from the
specimen and separate it by electrophoresis.
Bands produced are complementary mRNA
to the probe, and intensity of bands are
proportional to amount of specific mRNA.
17. 2.Ribonuclease protection
32Plabeled probe is hybridized in
solution with total RNA from specimen
Hybrids are digested with ribonucleases
(double-stranded ones will be protected
from digestion)
The mixture is separated
on a sequencing gel
Visualized radioactivity reveals a
range of intensity proportional to the
content of specific RNA in the sample.
18. 3.Reverse-transcription polymerase chain
reaction (RT-PCR)
Total RNA from sample is reverse-transcribed
into cDNA using reverse transcriptase.
Two oligonucleotides are added, chosen to
correspond to sequences in the target cDNA.
The sequence between the primers is amplified by
repeated cycles of synthesis, melting and hybridization.
The reaction mixture is run on a gel and the
DNA bands are visualized in the usual way.
The intensity of bands bears some relation to
the initial amount of mRNA in the sample.
19. Methods for DNA
1.In situ hybridization- designed to reveal the
spatial domains of gene expression in a
specimen.
An antisense probe is synthesized in vitro
complementary to the mRNA to be detected.
This is hybridized to the specimen and then
visualized.
Probes usually include extra chemical groups
recognizable by a commercially available
antibody for detection (e.g. digoxigenein or
DIG, a plant sterol)
Radioactive probes are used in radioactive in
situ hybridization (RISH), fluorescent dyes are
used in FISH.
20. 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
combined, which
allows the annealing
of complementary
DNA sequences.
21. In situ hybridization performed on a whole chick
embryo that have been fixed without being
sectioned. The probe used recognizes the mRNA
encoding Pax6 in the chick embryo. This probe is
labeled not with a radioactive isotope, but with a
modified UTP. To create this probe, a region of the
cloned Pax6 gene was transcribed into
mRNA, containing UTP conjugated with
digoxigenin. This does not interfere with the
coding properties of the resulting mRNA, but does
make it recognizably different from any other RNA
in the cell.
22. 2.Southern blot- to evaluate DNA extracts
from tissue samples.
This is a type of nucleic acid hybridization
test in which single-stranded DNA from two
sources interact.
Strands with similar nucleic acid sequences
will anneal by base pairing (A with T, and G
with C) to form double-stranded molecules.
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).
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23. Southern blotting. (1) DNA is treated with restriction
enzymes, and the resulting restriction fragments of DNA
are placed in a gel. (2) After the fragments of DNA are
separated on the gel by electrophoresis, the DNA is
denatured into single strands. (3) The 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.
The transfer buffer makes its way through the
gel, nitrocellulose paper, and towels by capillary
action, taking the DNA with it. The single-stranded DNA is
stopped by the nitrocellulose paper. (4) Blot is incubated
with radioactive or fluorescent probes in sealed bag. (5)
The positions of the DNA in the paper directly reflect the
positions of the DNA fragments in the gel.
24. *Blue dress of White House employee Monica Wilson
stained with semen of former US President Bill Clinton!
25. Southern blots (zoo blots) of various organisms. DNA using a
radioactive probe from the Antennapedia gene of Drosophila melanogaster.
Autoradiography shows that Drosophila genes contain several portions that
are like Antennapedia genes in structure and that many organisms contain
several genes that will hybridize this radioactive gene fragment, suggesting
that Antennapedia-like genes exist in these organisms. The numbers beside
the blots indicate size of bands, in kilobases.
26. 3.MICROARRAYS provide a means to measure &
monitor the expression of thousands of genes at once
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27. DNA microarray analysis
can reveal differences in
gene expression in yeast
cells under different
experimental conditions.
cDNA prepared from mRNA
isolated from wild-type
Saccharomyces cells grown on
glucose or ethanol is labeled
with different fluorescent dyes.
If a spot is , expression
of that gene is the same in
cells grown either on glucose
or ethanol. If a spot is ,
expression of that gene is
greater in cells grown in
glucose. If a spot is ,
expression of that gene is
greater in cells grown in
ethanol.
28. 3. GENE TARGETING (KNOCK-OUT) EXPERIMENTS-
wild type alleles are replaced with mutant ones.
There are 2 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)
29.
30. METHODS OF RECOMBINANT DNA
TECHNOLOGY
Knowledge of the molecular biology of cells
makes it possible to experimentally move from
gene to protein and from protein to gene!
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31. 1. Bacterial 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.
Because they can be used to create clones of genes,
plasmids are called CLONING VECTORS.
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......geneticsvideosrestriction.exe
32. Transfection-
cells are
incubated in
solution that
makes them
“drink” in
cloned DNA
usually mixed
with antibiotic
resistance
genes
33. Electropo
lation-
high-
voltage
pulse
“pushes”
the
cloned
DNA into
cell
34. Microinjection- cloned gene in
solution is injected into the cell
nucleus. The 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.
A “gene gun” 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.
35. Transposable 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.
Known as
jumping
genes since
they can
move about
on the
chromosome
or among
chromosomes.
36. Transgenic 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. Because a
transgene is integrated into
the recipient genome by non-
homologous
recombination, it does not
disrupt endogenous genes.
37. 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.
If the novel gene is present in these cells, the animal and
its stem cells can be used as a founder for breeding.
Such animal models allow researchers to test
therapeutic compounds and study the molecular basis of
given diseases.
Mouse disease models now exist for cystic fibrosis, beta-
thalassemia, atherosclerosis, retinoblastoma, and
Duchenne muscular dystrophy.
Somatic cells may also be grown in cell culture and
genetically modified by fusion with the enucleated egg.
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.
38. Isolating embryonic stem (ES) cells and incorporating them
into recipient embryos resulting into cells of different genetic
constitution appearing in the same organism (chimera). ES
cells treated as transgenics have been useful in determining
how genes are regulated during development of mice.
39. To analyze the role of BMP7 in
development, a bacterial gene for
neomycin resistance is inserted into
BMP7, destroying its ability to
function.
The mutant BMP7 genes are inserted
into neomycin sensitive ES
cells, heterozygous ES cells are then
microinjected into mouse
blastocysts, resulting to chimeras
which are then mated to wild-type
mice.
Heterozygous mice are
inbred, producing mutant mice which
lacked eyes and kidneys.
40. In the absence of the BMP7 protein, cells that
form the eyes & kidneys stop dividing and die.
Gene targeting makes transgenic mice that are
missing specific genes. In this way gene targeting
can be used to analyze the roles of particular
genes during mammalian development.
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41. Today, transgenic
s 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!
42. 4. ANTI- SENSE RNA-
interrupts translation
of mRNA to protein by
introducing single
strands of RNA
targeted to bind with
the mRNA
Generated by cloning
DNA into vectors with
promoters at both ends
of the inserted gene.
When incubated with
RNA polymerase and NTPs, the promoter will transcribe the
message in the wrong direction.
Transgenic 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. These tomatoes make only
10% of the normal amount of the enzyme.
43. The double-stranded
RNA molecules are
recognized by an RNase
and degraded into short
fragments.
This 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.
