Nucleic Acid Manipulations

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NUCLEIC ACIDMANIPULATIONS

Chapter Outline

Nucleic Acid Hybridization

The Polymerase Chain Reaction

Nucleic Acid Sequencing


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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


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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.


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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.


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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.


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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.


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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.


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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.


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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.


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Things to note:

Four components of DA

Hybridization

1. Target DNA

2. Probe

3. Detection System

4. Format


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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.


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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).


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Southern Hybridization Assays


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In Situ Hybridization Assays


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ENDReported by:

Jackquero V. Datinguinoo

BS Biology 4101

Nucleic Acid Manipulations

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