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TYPES OF DNA HYBRIDIZATION
Transcript of TYPES OF DNA HYBRIDIZATION
What is DNA hybridization.
Principle and basic procedure.
WHAT IS HYBRIDIZATION
Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing. In a reversal of this process, a double-stranded DNA (or RNA, or DNA/RNA) molecule can be heated to break the base pairing and separate the two strands. Hybridization is a part of many important laboratory techniques such as polymerase chain reaction and Southern blotting.
DNA HYBRIDIZATION procedure
DNA PROBE/GENE PROBE
Synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complimentary base pairing in a mixture of bio molecule.
MECHANISM OF DNA PROBE;
Based on Denaturation and renaturation
All blotting procedures begin with a standard process called gel electrophoresis when DNA, RNA, or proteins are loaded on to an agarose or acrylamide gel and separated on the gel through an electric field. Two types of gels are commonly used: agarose gels and acrylamide gels. Transfer is initiated when nitrocellulose or nylon membrane is laid on top of the gel and biological molecules are transfer from the gel to the membrane. Hybridization / blotting is a technique in which biological molecular (DNA, RNA or protein) are immobilized onto a nylon or nitrocellulose membrane.
Single stranded target DNA is bound to a membrane support
DNA probe labeled with detector substance is added
DNA probe pairs with the complimentary target DNA
Sequence of nucleotide in the target DNA can be identified
TYPES OF BLOTTING OR HYBRIDIZATION TECHNIQUE
SOUTHERN HYBRIDIZATION DNA-DNA
The technique was developed by E.M. Southern in 1975.
The Southern blot is used to detect the presence of a particular piece of DNA in a sample.
The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.
Later this technique is extended to RNA and protein(northern and western blotting)
Modified southern and northern blotting technique
1. BIOTECHNOLOGY BY U.SATHYANARAYANA
2. MICROBIOLOGY BY PRESSCOTT,HARLEY KLEIN
A probe (a piece of nucleic acid with identical and specific sequence to the organism or gene of interest) can then hybridize (join) to the biological molecules (DNA, RNA or protein) with identical sequence on the membrane. The hybridization between the blotted DNA and probe is visualized by labeling the probe in some way. Short fragments of DNA that have a nucleotide sequence complementary to the molecule being analyzed are normally used as probes in Southern and Northern blots. Antibodies that react with the protein being analyzed are used as probes in a Western blot.
The sample DNA(or RNA) from different individual are fragmented on to a nitrocellulose filter in the form of dots
The DNA is then denatured and then the filter is bached at 800c to fix the DNA firmly to the filter
The filter is pre treated to prevent non specific binding of the probe to the filter
The filter is then treated with appropriate radio active single stranded DNA probe under condition favoring hybridization
Filter is then washed repeatedly to remove the free probe
The hybridized probes are detected by auto radiography
Steps involved in southernblotting
1. Digest the DNA
with an appropriaterestriction enzyme.
2. Run the digest
on anagarose gel.
3. Denature the DNA
(usually while itis still on the gel).
4. Transfer thedenatured DNA to themembrane.
Traditionally, anitrocellulose membraneis used.
5.Probe the membranewith labeled ssDNA.
6.Visualize radioactivelylabeled target sequence.
Northern Blot: RNA-DNA*(RNA*)
Alwine adapted Southern's method for DNA to detect, size and quantify RNA – 1977.
No need to digest RNA with restriction enzymes.
Use formaldehyde to break H-bonds and denature RNA because single-stranded RNA will form intramolecular base pairs and "fold" on itself.
1. Isolate RNA & treat with formaldehyde.
2. Electrophorese RNA in denaturing agarose gel (has formaldehyde). Visualize RNA in gel using Ethidium bromide stain and photograph.
3. Transfer single-stranded RNA to nitrocellulose or nylon membrane. Covalently link RNA to membrane.
4. Incubate membrane (RNA immobilized on membrane) with labeled DNA or RNA probe with target sequence.
DNA on membrane.
Convert dsDNA to ssDNA.
Probe with DNA or RNA.
RNA on membrane.
No need to digest DNA.
Denature “folded” RNA with formaldehyde.
Probe with DNA or RNA
APPLICATION OF SOUTHERN HYBRIDIZATION
Invaluable method in gene analysis.
Important for the conformation of cloning result .
Useful for mapping restriction site around a single copy gene sequences.
Forensically applied to detect minute quantity of DNA to identify parenthood, thieves, rapist etc.
ZOO BLOTING: DNA pieces from one species can be used to detect DNA molecule from other related species.(e.g.: human to chimpanzee).
Northern blots are particularly useful for determining the conditions under which specific genes are being expressed, including which tissues in a complex organism express which of its genes at the mRNA level.