DNA HYBRIDIZATION ppt

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OVER VIEW:

OVER VIEW INTRODUCTION SOUTHERN HYBRIDIZATION NORTHERN HYBRIDIZATION WESTERN HYBRIDIZATION DOT HYBRIDIZATION COLONY HYBRIDIZATION REFERENCES

INTRODUCTION:

INTRODUCTION What is DNA hybridization. Principle and basic procedure. DNA probe Detector systems

WHAT IS HYBRIDIZATION:

WHAT IS HYBRIDIZATION Hybridization is the process of establishing a non-covalent , sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid

DNA HYBRIDIZATION:

DNA HYBRIDIZATION PRINCIPLE -Single stranded DNA molecule recognize and specifically bind to a complimentary DNA strand in a mixture of other DNA strand BASIC PROCEDURE - 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

DNA PROBE/GENE PROBE:

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

DETECTOR SYSTEM IN DNA HYBRIDIZATION:

DETECTOR SYSTEM IN DNA HYBRIDIZATION RADIO ACTIVE DETECTOR SYSTEM NON RADIO ACTIVE DETECTOR SYSTEM

RADIO ACTIVE DETECTOR SYSTEM:

RADIO ACTIVE DETECTOR SYSTEM ACGTTAGCA Target DNA TGCAATCGT Label DNA probe ACGTTAGCA TGCAATCGT

NON RADIO ACTIVE DETECTOR SYSTEM :

NON RADIO ACTIVE DETECTOR SYSTEM This detecting system is based on the enzymatic conversion of a chromogenic substrate or chemiluminiscent substrate. Biotin labeled nucleotide are incorporated in to the DNA probe.

Procedure for chemiluminiscent detection :

Procedure for chemiluminiscent detection Biotin labeled DNA probe is hybridized to the target DNA The egg white protein AVIDIN is added to bind biotin. Then biotin labeled enzyme such as alkaline phosphate is added to attach to avidin , this protein have four separate biotin binding site can bind to biotin labeled enzyme and biotin labeled DNA probe. On addition of a chemiluminiscent substrate , alkaline phosphate convert it to a light emitting substrate.

Procedure for chromogenic detection:

Procedure for chromogenic detection Here enzyme peroxidase is employed in place of alkaline phosphate , and when a chromogenic substrate is added color is produced and which can be measured

NUCLEIC ACID BLOTTING TECHNIQUE:

NUCLEIC ACID BLOTTING TECHNIQUE Blotting – refers to the process of immobilization of sample nucleic acid in solid support. The blotted nucleic acid are then used as target in the hybridization experiment for their specific detection

TYPES OF BLOTTING OR HYBRIDIZATION TECHNIQUE:

TYPES OF BLOTTING OR HYBRIDIZATION TECHNIQUE SOUTHERN BLOTTING NORTHERN BLOTTING DOT BLOTTING COLONY BLOTTING WESTERN BLOTTING

SOUTHERN BLOTTING/ SOUTHERN HYBRIDIZATION:

SOUTHERN BLOTTING/ SOUTHERN HYBRIDIZATION First developed by E.M. SOUTHERN. DNA-DNA hybridization is the basis Later this technique is extended to RNA and protein(northern and western blotting)

FACTORS AFFECTING SOUTHERN BLOTTING:

FACTORS AFFECTING SOUTHERN BLOTTING STRINGENT CONDITION : Refers to the specificity with which a particular DNA target sequence is detected by a probe . Thus with high stringent condition only complete compliment sequence will bind . Low stringent condition will allow hybridization of partially matched sequence . Therefore stringency is very essential in southern blotting technique

2. MEMBRANE FOR BLOT TRANSFER:

2. MEMBRANE FOR BLOT TRANSFER Early years nitrocellulose was used but not using now a days because it is fragile and has to be handled with care . Now NYLON MEMBRANES are using as they possess high tensile strength and better binding capacity for nucleic acid.

APPLICATION OF SOUTHERN HYBRIDIZATION:

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 BLOTTING/ NORTHERN HYBRIDIZATION:

NORTHERN BLOTTING/ NORTHERN HYBRIDIZATION Technique for the specific identification of RNA It is an extension method of southern blotting

ADVANTAGES:

ADVANTAGES Useful in the identification and separation of RNA that is complimentary to a specific DNA probe Sensitive test for the detection of transcription of a DNA sequence that is used as probe DISADVANTAGES This is not really practicable since each gene may give rise to two or more RNA transcripts

WESTERN BLOTTING:

WESTERN BLOTTING Involves the identification of protein. Useful to understand nucleic acid function particularly during the course of gene manipulation

PROCEDURE:

PROCEDURE Protein bands are separated by poly acrilamide gel electrophoresis. Protein bands transferred on to a nylon membrane or nitro cellulose. The specific bands are identified in a variety of ways such as Antibodies are commonly used as probe for detection of specific antigen. Lecithin are also used as probes for identification of glycoprotein

DOT BLOTTING:

DOT BLOTTING Modified southern and northern blotting technique. PROCEDURE 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 80 0 c 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

COLONY AND PLAQUE BLOTTING:

COLONY AND PLAQUE BLOTTING Used for identification and purification of colonies PROCEDURE Bacteria are grown as colonies on an agar plate Nitrocellulose filter paper over laid on the agar plate They are permanently fixed by heat Then treatment with alkali the cell lyses and get de natured When DNA prints are exposed to specific probes , hybridization occur. Hybridization complex can be localized and detected by autoradiography

REFERENCES:

REFERENCES BIOTECHNOLOGY BY U.SATHYANARAYANA P.N:97-100,173-175 2. MICROBIOLOGY BY PRESSCOTT,HARLEY KLEIN P.N:419-420

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DNA hybridization Nucleic acids from different species can form hybrids Can be used to detect similar DNA sequences in two different species or within the genome of a single species

Western blotting:

Western blotting

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