Nucleic acid hybridization

hybridization - hybridization probes - types of hybridization and applications

1. NUCLEIC ACID HYBRIDIZATION ThangaMallika.K II M.Sc., Microbiology 18PY20

2. INTRODUCTION: A basic technique in molecular biology. in which single stranded Nucleic acids are allowed to interact so that complexes called HYBRIDS are formed by molecules with similar complementary sequences.

3. NUCLEIC ACID HYBRIDIZATION : A technique which has the ability of indivudial single stranded nucleic acid molecules to form double stranded molecules. The principle of hybridization is the addition of a probe to a complex mixture of target DNA. The mixture is incubated under conditions that promote the formation of hydrogen bonds between complementary strands.

4. ABOUT DNA: Watson and Crick model of DNA was followed and it possess two strands which runs in antiparallel directions and those strands are held by hydrogen bonds. According to Watson-Crick base pairing, Adenine pairs with Thymine and Guanine pairs with Cytosine by hydrogen bonding. The mechanism can be achieved by means of Denaturation and Annealing.

5. Since, Nucleic acid hybridization is a process used to identify specific DNA sequences. This is performed by means of DNA probes. Probes used in hybridization reactions are usually chemically synthesized DNA or RNA that has been labelled with fluorescent dye or radioactive isotope. Nucleic acid hybridization can be done in all combinations: DNA-RNA, DNA-DNA or RNA-RNA, Insitu Hybridization and FISH analysis.

6. FACTORS AFFECTING NUCLEIC ACID HYBRIDIZATION: Strand length Base Composition Chemical Environment.

7. HYBRIDIZATION PROBES: It is a nucleic acid fragment that is complementary to another nucleic acid sequence and thus, when labeled (with radioisotope, fluorescent dye, etc.) can be used to identify complementary segments. A probe actually hybridizes to single stranded nucleic acid (DNA/RNA) molecules because of complementarity between the probe and target. Nucleic acid probes can be synthesized in the laboratory, as single and double stranded probes, but a working nucleic acid should be a single stranded only to bind with complementary target (sequence).

8. Probes are of three types: DNA probes : it is a short sequence of DNA labeled isotopically or chemically that is used for the detection of a complementary nucleotide sequences. RNA probes: it is a short sequence of RNA labeled isotopically or chemically that is used for the detection of a complementary nucleotide sequences. They are also known as riboprobes or complementary probes and are often used in insitu hybridization because of high sensitivity.

9. Oligonucleotide probes: it is a short sequence of nucleotides synthesized to match a region where a mutation is known to occur and then used as a molecular probe to detect the mutation.

10. LABELLING OF PROBES: Hybridization probes can be labelled by two methods: 1. Invivo Labelling 2. Invitro Labelling INVIVO LABELLING : By supplying labeled nucleotides to the cultured cells. INVITRO LABELLING : An enzyme is used to incorporate a labeled nucleotide in the probe.

11. TYPES OF HYBRIDIZATION: There are mainly three techniques of hybridization. They are as follows: 1. Southern Hybridization 2. Northern Hybridization 3. Colony Hybridization

12. SOUTHERN HYBRIDIZATION : Southern blot is a techniques employed for detection of a specific DNA sequence in DNA samples that are complementary to a given RNA or DNA sequence. It was first given by E.M Southern, a British biologist. This methods includes separation of restricted DNA fragments by electrophorosis and then transferred to a nitrocellulose or a nylon membrane, followed by detection of the fragment using probe hybridization.

13. APPLICATIONS: Southern blots are used in gene discovery, mapping, evolution and development studies. To identify specific DNA in the sample. To isolate desired DNA for construction of DNA. Used in phylogenic analysis. Used to make RFLP maps.

14. NORTHERN HYBRIDIZATION: Northern blotting was developed by James Alwine , George stark and David Kemp (1977). In this technique, RNA is being analysed instead of DNA. It is a technique by which RNA fragments are separated by electrophorosis and immobilized on a membrane . The identification of specific RNA is done by using nucleic acid probes. It helps to study gene expression by detection of RNA.

15. APPLICATIONS: To study the gene expression of various tissues, organs, development stages, pathogen infections. mRNA splicing studies. Identification of transferred genes in transgenic individuals.

16. COLONY HYBRIDIZATION : It is a rapid method of isolating a colony containing a plasmid harboring a particular sequence or a gene from a mixed population. The colonies to be screened are first replica plated onto a nitrocellulose filter disc that has been placed on the surface of an agar plate prior to inoculation.

17. In situ Hybridization : It is a technique that employs a labeled complementary nucleotide strand for localizing specific DNA or RNA sequence targets within fixed tissues and cells. There are two ways to detect DNA or RNA targets: i ) Chromogenic insitu hybridization ii) Flourescence insitu hybridization.

18. CISH FISH

19. APPLICATIONS: Library screening Southern blot Northern blot ASOs ( Allele-specific oligonucleotides ) to detect mutations.