Dna probes

To describe about DNA probes and preparation of radio labeled and non- radio labeled DNA probes.

1. DNA probes Presented by Fathima Hameed

2. Outlines Introduction Method of nucleic acid hybridization Uses of DNA probes Features of DNA probes Labeling of DNA Methods of labeling DNA preparation of labeled nucleotides Uses of end labeled DNA Detection of probes

3. introduction probes are short section of DNA or RNA with an additional tagged or labeled chemically entity that are used to bind it’s Complimentary strand and there by allows detection of candidate Nucleic acid molecules.

4. The chemically synthesized entity are; fluorescent molecule an attachment to a colored bead quantum dots photo chromic compounds isotopic labeling non- isotopic labeling It allows us to visualize when a probe attaches to DNA, RNA or other target nucleic acids.

5. For hybridization, it involves interaction of single strands of two sources of nucleic acids;

6. Gene probe: it generally longer than 500 bases . it consist most of a target gene . Hybridization probes: probe is labeled in “standard hybridization assay ” target is labeled in “reverse hybridization assays ” nucleic acid probes can be single or double standard . working probe must be single stranded.

7. Method of nucleic acid hybridization

8. features Hybridization probe type DNA origin cell based DNA cloning or PCR characteristics of starting material Normally double stranded, 0.1 kb to hundreds of kb for conventional DNA clones, 0.1 kb to > 20 kb for PCR products labeling Usually by DNA polymerase based DNA strand synthesis

9. Labeling of DNA Probes can be labeled at specific location within the oligonucleotides Some probes are of defined length . Some probes are heterogeneous population of labeled molecules. two ways of labeling; In vivo labeling In vitro labeling

10. In vivo labeling: DNA and RNA can be directly labeled inside tissue culture cells by adding labeled deoxynucleotides in culture plate in vivo . This method is restricted only to prepare labeled viral DNA from virus infected cells and to study RNA Processing events .

11. In vitro labeling: It is a more versatile . It involving in vitro labeling of purified RNA,DNA or Oligonucleotide using DNA polymerase for incorporation of labeled nucleotides.

12. In vitro labeling of DNA can be done by various methods as follows ,

13. 1.DNA nick translation It involves insertion of random single strand breaks called “nicks ”. The nicks in one strands of double stranded target DNA which exposes 3’- OH termini and 5’-PO4 termini . The nicks are introduced by endonuclease like pancreatic deoxyribonuclease I (DNase I ). enzymes are used for nick translation, Dnase I E.coli DNA polymerase I

14. Dnase I – exonuclease DNA polymerase- multi subunit enzyme The exonuclease attacks the 5’ termini of a nick & removes the nucleotides in 5’→3’ direction . DNA polymerase adds the nucleotides to the free 3’- OH group, in 5’→3’ direction.

15. Advantages: This method requires 100-fold less radioactive precursor . The amount of radio labeled incorporated depends on number of nicks created by Dnase I. Disadvantages: Only one complete regeneration is takes place . Reaction does not proceed further.

16. 2.Random primed DNA labeling This method is known as “ oligo -labeling ” It is based upon hybridization of a mixture of all possible hexanucleotides . The template DNA is initially denatured . The synthesis of new complementary DNA strands is primed by bound hexanucleotides . Random hexanucleotides to bind at complementary sequences at which extension takes place through PCR . It was catalyzed by klenow subunit of DNA polymerase I.

17. Random primed DNA labeling

18. Advantages: It produce labeled DNA’s of high specific activity . Primer represents all possible sequence combination & uniform labeling of DNA occurs . Random priming is inherently simpler than nick translation, because the requirements for two nuclease activity are eliminated . The probes generated by random priming method are more homogenous in size . Probes are more reproducibly in hybridization reactions.

19. Disadvantages: Since binding of primer to template DNA is only random . The length of random primers is crucial, primers shorter than 6 bases are very poor primers.

20. 3.PCR mediated DNA labeling The standard PCR reaction can be modified to incorporate labeled nucleotides . This method commonly using in 2 ways, Standard PCR based DNA labeling Primer mediated 5’ end labeling

21. Standard PCR based DNA labeling

22. Standard PCR based DNA labeling: The probe generation reaction is modified to incorporate one or more labeled nucleotide precursors at a concentration same as oligonucleotide concentrations. Primer mediated 5’ end labeling: Radio labeled probes can be generated for both strands using equal concentration of primers or heavily in favor of one strand of DNA using higher concentration of one primer. It uses a 5’ end labeled primers.

23. Advantages: Defined segment of target DNA can be amplified independently of restriction sites . Amount of template DNA is required very small . No need to isolate fragments of DNA or to sub-clone into vectors containing bacteriophage promoters.

24. Preparation of labeled nucleotides

25. Isotopic labeling: They can be detected directly in solutions or on x-ray film using autoradiography . The strength of autoradiography signals depends on intensity of radiation emitted by radioisotope and duration of exposure . Mostly using isotopes are 32P, 33P,35S& 3H. Radioisotopes Half-life Energy of emission 3H 12.4 years 0.019 MeV 32P 14.3 years 1.710 MeV 33P 25.5 years 0.248 MeV 35S 87.4 years 0.167 MeV

26. Non-isotopic labeling: In this systems involved the use of non radioactive probes . Two types of non radioactive labeling are conducted, Direct non-isotopic labeling Indirect non-isotopic labeling

27. Direct non-isotopic labeling: Where a nucleotide containing label such as, Fluorescein Texas red Rhodamine These will be detected when incorporated with the help of spacer molecule . These modified nucleotides having tag & fluoresce when excited by light of certain wavelength.

28. Indirect non-isotopic labeling: It involves chemical linkage of reporter molecule to a nucleotide . When this modified nucleotide is incorporated into DNA, then it is specifically bound to a protein or other ligand . It has high affinity against the reporter group . Long spacer is introduced between nucleotide and reporter .

29. Indirect non-isotopic labeling

30. Two widely used non-isotopic labeling methods are,

31. End labeled DNA can be used as; Molecular weight standards in southern blotting . Probes in gel retardation experiments . Traces for small quantities of DNA’s on gels . Probes for screening bacterial colonies or plaques . Substrates for Maxam-Gilbert sequencing . Probes for RNA mapping with S1 nuclease or mung bean nuclease. primers in primer –extension reactions .

32. Detection of non-radioactively labeled probes after hybridization Affinity molecules are conjugated with a variety of marker groups or molecules . They include various fluorophores or enzymes such as alkaline phosphatase and peroxidase which can permit detection via, colorimetric assays fluorescent assays chemiluminescence

33. Thank you