Nucleic Acid Manipulations
-
Upload
orekaj-dattebayo -
Category
Documents
-
view
224 -
download
0
Transcript of Nucleic Acid Manipulations
-
8/8/2019 Nucleic Acid Manipulations
1/19
NUCLEIC ACIDMANIPULATIONS
Chapter Outline
Nucleic Acid Hybridization
The Polymerase Chain Reaction
Nucleic Acid Sequencing
-
8/8/2019 Nucleic Acid Manipulations
2/19
Hybridization
has emerged form the development in the
area of genetic engineering and molecular
biology.
this technique is used to detect the presenceof DNA from pathogens in clinical specimens
and locate specific genes in cell.
is a method for identifying closely related
nucleic acid molecules within twopopulations, a complex target population and
a comparatively homogeneous probe
population
-
8/8/2019 Nucleic Acid Manipulations
3/19
Principles of Nucleic Acid
Hybridization
Definition of nucleic acid hybridization
Nucleic acid hybridization involves mixing
single strands of two sources of nucleic acids,
a probe which typically consists of a
homogeneous population of identified
molecules (e.g. cloned DNA or chemically
synthesized oligonucleotides) and
a target which typically consists of a complex,
heterogeneous population of nucleic acid
molecules.
-
8/8/2019 Nucleic Acid Manipulations
4/19
If either the probe or the target is initially
double-stranded, the individual strands must be
separated, generally by heating or by alkaline
treatment. After mixing single strands of probewith single strands of target, strands with
complementary base sequences can be allowed
to reassociate. Complementary probe strands
can reanneal to form homoduplexes, as cancomplementary target DNA strands.
-
8/8/2019 Nucleic Acid Manipulations
5/19
However, it is the annealing of a probe DNA
strand and a complementary target DNA strand
to form a labeled probe-target heteroduplex that
defines the usefulness of a nucleic
acid hybridization assay. The rationale of
the hybridization assay is to use the
identified probe to query the target DNA by
identifying fragments in the complex target
which may be related in sequence to the probe.
-
8/8/2019 Nucleic Acid Manipulations
6/19
-
8/8/2019 Nucleic Acid Manipulations
7/19
-
8/8/2019 Nucleic Acid Manipulations
8/19
Assay
1. DNA from a virus or cell is denatured with alkali
to separate the strands.
2. The single strands of DNA are then attached to
a solid support such as nitrocellulose or nylon
membrane so that the strands do not reanneal.3. The DNA is attached to the membrane by its
sugar-phosphate backbone with the nitrogenous
bases projecting outward.
4. To characterized or identify the target DNA, asingle-stranded DNA or RNA molecule of
known origin called a probe is added to the
membrane in a buffered solution.
-
8/8/2019 Nucleic Acid Manipulations
9/19
More about the probe:
this allows the formation of hydrogen bonds
between complementary bases.
the probe is so called because if it used to
seek or probe for DNA sequences, islabelled with a reporter group, which may be
a radioactive atom or an enzyme whose
presence can be easily detected.
the probe is allowed to react with the targetDNA; then any unreacted probe is removed
by washing in buffered solutions.
-
8/8/2019 Nucleic Acid Manipulations
10/19
contd
after the washes, all that remains on the
nitrocellulose is the target DNA and any
probe molecules that have attached to
complementary sequences in the target
DNA, forming stable hybrids.
-
8/8/2019 Nucleic Acid Manipulations
11/19
DNA Hybridization Assay
continued...5. Hybridization of target and probe DNAs is
detected by assaying for the probes reporter
group.
6. If the reporter group is detected, hybridization
has taken place. If no reporter group is detected,
it can be assumed that the target molecule does
not have sequences that are complementary tothose of the probe and hence sequences that are
complementary to those of the probe and hence
the gene or DNA segment sought is not present
in the sample.
-
8/8/2019 Nucleic Acid Manipulations
12/19
Things to note:
Four components of DA
Hybridization
1. Target DNA
2. Probe
3. Detection System
4. Format
-
8/8/2019 Nucleic Acid Manipulations
13/19
Three Common Formats in Solid
Phase Hybridization Assays1. Dot Blot Assay - a specified volume of
sample or specimen is spotted onto a small
area of nitocellulose membrane.2. Southern Hybridization Assays invlove
restriction enzyme digestion and agarose
gel electrophoresis of the target DNA prior
to the hybridization assay.- the different bands on the agarose gel aretransferred by capillary action onto a nitocellulose
or nylon membrane in a blot apparatus.
-
8/8/2019 Nucleic Acid Manipulations
14/19
-
8/8/2019 Nucleic Acid Manipulations
15/19
Dot blot hybridization analysis of DNA from the LMW and HMW
gel fractions with probes for repetitive sequences known to be present
in T.brucei: subtelomeric repeats (subtel), the conserved second part
of VSG genes (VSG), 70, 177 and 50 bp and rDNA repeats and
kDNA minicircles; hybridization signals are quantitated relative to
non-fractionated control DNA (bloodstream form DNA digest;
tubulin is 1).
-
8/8/2019 Nucleic Acid Manipulations
16/19
Southern Hybridization Assays
-
8/8/2019 Nucleic Acid Manipulations
17/19
In Situ Hybridization Assays
-
8/8/2019 Nucleic Acid Manipulations
18/19
-
8/8/2019 Nucleic Acid Manipulations
19/19
ENDReported by:
Jackquero V. Datinguinoo
BS Biology 4101