Hybridization presentation
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Hybridization is a process where two complementary strands of nucleic acids interact to form a hybrid molecule. It involves denaturing double-stranded nucleic acids into single strands, then allowing complementary single strands to renature. Probes are single-stranded DNA or RNA fragments labeled with a detectable tracer. Target nucleic acids are denatured and mixed with probes, and hybridization detected using the probe label if complementary sequences are present. Common applications include identifying transgenic genes and determining protein concentrations.
Hybridization presentation
- 1. HYBRIDIZATION BY-SANJU SAH ST. XAVIER’S COLLEGE, MAITIGHAR, KATHMANDU, NEPAL DEPARTMENT OF MICROBIOLOGY (BATCH-2017)
- 2. HYBRIDIZATION? HYBRIDIZATION IS A PROCESS OF ESTABLISHING NON COVALENT, SEQUENCE SPECIFIC INTERACTION BETWEEN TWO OR MORE COMPLEMENTARY STRANDS OF NUCLEIC ACIDS INTO A SINGLE HYBRID. IN SIMPLE WORDS,IT IS A MOLECULAR TECHNOLOGICAL PROCESS IN WHICH TWO SINGLE- STRANDED NUCLEIC ACID MOLECULES WITH COMPLEMENTARY BASE SEQUENCES FORM A DOUBLE-STRANDED NUCLEIC ACID MOLECULE. NUCLEIC ACID HYBRIDIZATION TECHNOLOGY IS A FUNDAMENTAL TOOL IN MOLECULAR BIOLOGY, AND HAS BEEN APPLIED IN VARIOUS FIELDS SUCH AS :
- 3. PRINCIPLE : THE TECHNIQUE OF NUCLEIC ACID HYBRIDIZATION IS ESTABLISHED AND DEVELOPED ON THE BASIS OF THE DENATURATION AND RENATURATION OF NUCLEIC ACIDS. HYDROGEN BONDS IN DOUBLE STRANDED NUCLEIC ACIDS CAN BE DISRUPTED BY SOME PHYSICOCHEMICAL ELEMENTS, AND TWO STRANDS OF NUCLEIC ACIDS ARE SEPARATED INTO SINGLE STRAND. THEN THESE SINGLE STRANDED DNA MOLECULES (PROBES) RECOGNIZE AND SPECIFICALLY BIND TO A COMPLEMENTARY DNA STRAND IN A MIXTURE OF OTHER DNA STRAND.
- 6. IF DIFFERENT SINGLE-STRANDED DNA MOLECULES, OR DNA AND RNA MOLECULES, OR RNA MOLECULES ARE MIXED TOGETHER IN A SOLUTION, AND THE RENATURATION IS ALLOWED TO OCCUR UNDER PROPER CONDITIONS, SINGLESTRANDED DNA OR RNA WILL BIND WITH EACH OTHER TO FORM A LOCAL OR WHOLE MOLECULE OF DOUBLE-STRANDED STRUCTURE AS LONG AS THE SINGLE-STRANDED MOLECULES ARE COMPLEMENTARY, NO MATTER WHAT KIND OF SOURCES THEY COME FROM. NUCLEIC ACID HYBRIDIZATION AS A TECHNIQUE INVOLVES USING A LABELED NUCLEIC ACID PROBE, WHICH IS A KNOWN DNA OR RNA FRAGMENT. A PROBE LABELED WITH DETECTABLE TRACER IS THE PREREQUISITE FOR DETERMINING A SPECIFIC DNA SEQUENCE OR GENE IN A SAMPLE OR GENOMIC DNA BY NUCLEIC ACID HYBRIDIZATION.
- 7. THE TARGET NUCLEIC ACIDS TO BE ANALYZED ARE USUALLY DENATURED, AND THEN MIXED WITH THE LABELED PROBE IN THE HYBRIDIZATION SYSTEM. THE PROBE WILL BIND TO THE SEGMENT OF NUCLEIC ACID WITH COMPLEMENTARY SEQUENCE UNDER PROPER CONDITIONS. THE HYBRIDIZATION CAN BE IDENTIFIED BY THE DETECTION OF THE TRACER LABELING THE PROBE. THUS THE EXISTENCE OR THE EXPRESSION OF SPECIFIC GENE CAN BE DETERMINED. PREPARATION OF PROBES : PROBES MAY BE SINGLE- STRANDED OR DOUBLE STRANDED MOLECULES, BUT THE WORKING PROBE MUST BE SINGLE-STRANDED MOLECULES. THE PROBES USED IN HYBRIDIZATION
- 8. GENOMIC DNA PROBES CAN BE PREPARED FROM THE CLONED DNA FRAGMENT IN PLASMID. CDNA PROBES CAN BE PREPARED FROM THE CLONED CDNA IN PLASMID, OR AMPLIFIED DIRECTLY FROM MRNA BY RT-PCR. RNA PROBES ARE USUALLY TRANSCRIBED IN VITRO FROM A CLONED CDNA IN A PROPER VECTOR. THE SIZE OF GENOMIC DNA PROBES, CDNA PROBES AND RNA PROBES MAY BE 0.1 KB TO 1 KB. • LABELING OF PROBES PROBE IS USUALLY LABELED WITH A DETECTABLE TRACER, WHICH IS EITHER FLUORESCENT DYES OR RADIOACTIVE ISOTOPES. THE PURIFIED OLIGONUCLEOTIDE CAN ALSO BE LABELED IN VITRO USING A SUITABLE ENZYME TO ADD THE LABELLED NUCLEOTIDE TO THE END OF THE OLIGONUCLEOTIDE.
- 9. DETECTOR SYSTEM IN DNA HYBRIDIZATION: • RADIOACTIVE DETECTOR SYSTEM: AUTORADIOGRAPHY FOR RADIOACTIVE ISOTOPES. • NON-RADIOACTIVE DETECTOR SYSTEM : BASED ON THE ENZYMATIC CONVERSION OF A CHROMOGENIC SUBSTRATE OR CHEMILUMINISCENT SUBSTRATE. BIOTIN LABELED NUCLEOTIDE ARE INCORPORATED INTO THE DNA PROBE.
- 10. NUCLEIC ACID BLOTTING TECHNIQUE: BLOTTING REFERS TO PROCESS OF IMMOBILIZATION OF SAMPLE NUCLEIC ACID IN SOLID SUPPORT. THE BLOTTED NUCLEIC ACIDS ARE THEN USED AS TARGET IN THE HYBRIDIZATION EXPERIMENT FOR THEIR SPECIFIC DETECTION. TYPES OF BLOTTING TECHNIQUES: • SOUTHERN BLOTTING • NORTHERN BLOTTING • WESTERN BLOTTING • COLONY BLOTTING • DOT BLOTTING
- 12. ADVANTAGES OF DOT BLOT: • NO ELECTROPHORESIS REQUIRED AS SAMPLES ARE DIRECTLY LOADED ON NITROCELLULOSE MEMBRANE. • NO TRANSFER OF DNA FROM AGAR TO MEMBRANE REQUIRED. • DIFFERENT SAMPLES CAN BE RUN AT THE SAME TIME. DISADVANTAGES: • NO IDEA ON SIZE AND SHAPE OF NUCLEIC ACID OR PROTEIN IS ACCESSIBLE. APPLICATIONS: • TO IDENTIFY TRANSGENIC GENES. • TO DETERMINE CONCENTRATION OF PROTEIN IN SAMPLES.