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Lecture 5 dna finger, foot printing rflp
This document discusses DNA fingerprinting techniques including restriction fragment length polymorphism (RFLP) and DNA footprinting. RFLP involves using restriction enzymes to cut DNA at specific sites, resulting in fragments of varying lengths that can be used to differentiate individuals. DNA footprinting identifies the specific binding sites of DNA-binding proteins by detecting regions of DNA that are protected from cleavage by bound proteins. The document provides detailed explanations of the principles and procedures of these techniques.
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Lecture 5 dna finger, foot printing rflp
- 1. Dr. Vishnu Kumar Professor,Department of Biochemistry, SRMSIMS, Bareilly [email protected] [email protected] DNA Finger & DNA Foot Printing Restriction fragment length polymorphism ( RFLP)
- 2. Learning Objectives After completionof this lecture learner should be able to define : DNA Finger Printing Restriction fragment length polymorphism ( RFLP) Applications of RFLP DNA Foot Printing
- 3. Restriction fragment length polymorphism( RFLP) • Traditionally the most suitable method to detect a criminal in the scene of crime is matching of finger prints. With discovery of RDT a more useful tool is now RFLP or DNA finger printing. • Polymorphism Genetic diversity in a population is called polymorphism.
- 4. RFLP • It impliesthat the DNA sequence of one individual differs from that of the other. Some of these changes may affect the recognition site of RE. So the size of the restriction fragments will be different in different individual if their genome is subjected to the same RE. Such variation in the pattern of restriction fragments in the population is referred to as RFLP.
- 5. Restriction Fragment LengthPolymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be examined by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme.
- 6. Seven steps tounderstanding DNA fingerprinting: 1. Extracting the DNA from cells. 2. Cutting up the DNA using an enzyme. 3. Separating the DNA fragments on a gel. 4. Transferring the DNA onto paper. 5. Adding the radioactive probe. 6. Setting up the X-ray film. 7. Yes - we've got the result!
- 7. Any mutation ofa single nucleotide may destroy (CTGCAC or CTTAAC for HindII) and alter the length of the corresponding fragment. The term polymorphism refers to the slight differences between individuals, in base pair sequences of common genes. or A polymorphism is a clinically harmless DNA variation that does not affect the phenotype. RFLP analysis is the detection of the change in the length of the restriction fragments.
- 9. Isolating DNA isthe first step for many DNA-based technologies. DNA is found either in nuclear chromosomes or in organelles (mitochondria and chloroplasts). To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane, and so appropriately separate the DNA from other cell components. When doing so, care must be taken to ensure the process does not damage the DNA molecule and that it is recovered in the form of a long thread.
- 10. Extracted DNA isdigested with specific, carefully chosen, restriction enzymes. Each restriction enzyme, under appropriate conditions, will recognize and cut DNA in a predictable way, resulting in a reproducible set of DNA fragments (‘restriction fragments’) of different lengths. The millions of restriction fragments produced are commonly separated by electrophoresis on agarose gels. Because the fragments would be seen as a continuous ‘smear’ if stained with ethidium bromide, staining alone cannot detect the polymorphisms. Hybridisation must therefore be used to detect specific fragments.
- 11. DNA transfer iscalled ‘Southern blotting’, after E.M. Southern (1975), who invented the technique. In this method, the gel is first denatured in a basic solution and placed in a tray. A porous nylon or nitrocellulose membrane is laid over the gel, and the whole weighted down. All the DNA restriction fragments in the gel transfer as single strands by capillary action to the membrane. All fragments retain the same pattern on the membrane as on the gel.
- 12. Where conditionsare highly stringent, hybridisation with distantly related or non-homologous DNA does not happen. Thus, the DNA probe picks up sequences that are complementary and 'ideally‘ homologous to itself among the thousands or millions of undetected fragments that migrate through the gel. Desired fragments may be detected after simultaneous exposure of the hybridized membrane and a photographic film.
- 13. RFLP Technology Pictures Afteragarose has been poured into the gel mould, combs are immediately inserted to form wells and left until the gel hardens. The combs are then removed and the gel placed in an electrophoresis chamber.
- 14. Samples of digested DNA, withbromophenol blue dye added, are loaded into the wells with a pipette.
- 15. After electrophoresis, the gelis treated with NaCl to break the DNA double helix bonds and make it single-stranded. This allows later hybridisation with a single-stranded DNA probe.
- 16. The blotting trayis first prepared by saturating sponges with NaOH. Safety glasses and gloves are required, and a laboratory coat recommended.
- 17. Absorbent paper isplaced on top of the sponges to prevent direct contact with the gel.
- 18. Bubbles between theabsorbent paper and sponges are removed by rolling a pipette or a glass rod across the paper. This ensures a complete transfer of the solution all through the gel.
- 19. The treated agarosegel is placed on top of the absorbent paper.
- 20. Membrane is cut intothe appropriate size. The membrane is placed on top of the gel, then covered with a piece of absorbent paper.
- 21. The entire set-upis topped with a weight (here, a bottle of water standing on a piece of glass) to promote good transfer. After some hours the transfer is complete, the blotting paper is taken away, and the membrane stored until hybridisation with the probe.
- 22. The process ofhybridisation begins. A DNA is boiled to denature it to single strands.
- 23. The labeled probeis added to the container with the hybridisation solution and membrane, and incubated overnight in an oven. The following day, the membrane is removed from the hybridisation set up, and washed with the appropriate stringency solution.
- 24. The membrane isthen blotted dry and put into a cassette for holding X- ray film. The cassette is wrapped, or sealed with tape, and stored in a freezer until the film is sufficiently exposed, usually 1 to 4 days.
- 25. Clinical application ofRFLP. • 1) Diagnosis of genetic diseases e.g. sickle cell anemia. • 2) Forensic medicine e.g. identifying a criminal. • 3) Tracing of chromosome from parent s to offspring. e.g. paternity test.
- 27. 4. RFLPs canbe used determine the disease status of an individual. (e.g. it can be used in the detection of mutations particularly known mutations). 5. In human population genetics, geographical isolates and comparison of genetical makeup of related species.
- 28. Highly robust methodologywith good transferability between laboratories. No sequence information required. highly recommended for phylogenetic analysis between related species. Well suited for constructing genetic linkage maps. Simplicity—given the availability of suitable probes, the technique can readily be applied to any plant.
- 29. Large amounts ofDNA required. Automation not possible. Low levels of polymorphism in some species. Time consuming, especially with single- copy probes Costly. Moderately demanding technically.
- 30.
- 31. INTRODUCTION • DNA footprintingis a method of investigating the sequence specific of DNA – binding protein in vitro. • This technique can be used to study protein – DNA interactions both outside and within cell. • Techniques like DNA footprinting help elucidate which protein bind to these associated region of DNA and unravel the complexities of transcriptional control.
- 32. HISTORY • In 1978,Davidgalas and Albert Schmitz developed the DNA footprinting technique to study the binding specificity of the lac repressor protein . • It was originally a modification of the maxam – gilbert chemical sequencing technique .
- 33. PRINCIPLE • In thistechnique, nucleases like DNase I is used which will degrade DNA molecule. • Nucleases cannot degrade DNA if it is bounded by a protein. Thus that region is protected from degradation by nucleases. • This protected DNA region is called the foot print.
- 35. METHOD The simplest applicationof this technique is to assess whether a given protein binds to a region of interest within a DNA molecule •Polymerase chain reaction(PCR) amplify and label region if interest that contain a potential protein- binding site. •Add protein of interest to a portion of the labelled template DNA ; a portion should remain separate without protein ,for later comparison •Now, add a cleavage agent to both the portion of DNA template (cleavage agent is a chemical or enzyme that will cut at random locations in a sequence independent manner).
- 36. • Run boththe samples side by side on a polyacrylamide gel electrophoresis . • The portion of DNA template without protein bill will be cut at random locations , and thus when it is run on a gel ,will produce a ladder- like distributions. • The DNA template with a protein will result in ladder distribution with a break in it , the “footprint” , where the DNA has been protected from the cleavage agent .
- 37. Labeling • The DNAtemplate can be labeled at the 3’ or 5’ end , depending on the location of binding sites . • Labels that can be used are : radioactivity and fluorescence . • Radioactivity has been traditionally used to label DNA fragments for footprinting analysis. • Radioactive labelling is very sensitive and is optimal for visualising small amount of DNA . • Fluorescence is a desirable advancement due to the hazards of using radio-chemicals .
- 38. • However, ithas been difficult to optimize because it is not always sensitive enough to detect the low concentrations of the target DNA strands used in a DNA footprintings experiments. • Electrophoretic sequencing gels or capillary electrophoresis have been successfully in analysing foot printing of fluorescent tagged fragments .
- 39. Cleavageagent • A varietyof cleavage agent can be chosen . • Ideally a desirable agent is one that is sequence neutral , easy to use , and is easy to control . • The following cleavage agent are described in detail : DNase I is a large protein that function as a double – strand endonuclease . • It binds the minor group of DNA and cleaves the phospodiester backbone .it is good cleavage agent for footprinting because its size makes it easily physically hindered .thus is more likely to have to its action blocked by a
- 40. • Hydroxyl radicalsare created from the Fenton reaction , which involves reducing Fe2+ with H2o2 to form free hydroxyl molecules .These hydroxyl molecules react with the DNA backbone resulting in a break. Due to their small size , the resulting DNA footprint has a high resolution . • Ultraviolet irradiation can be used to excite nucleic acid and create photoreactions , which result in damaged bases in a DNA strand. Advantages of uv are that it reacts with very quickly and therefore capture the interactions
- 41. Applications • DNA footprinting is often used to identify the binding sites of proteins in a DNA molecule. • Researchers often use this technique to identify whether a particular protein can activate or inhibit transcription. • In addition, scientists also use this method to detect where proteins bind to DNA in a living cell.
- 42. LONG ANSWER QUESTIONS 1.Describe the Restriction fragment length polymorphism ( RFLP) 2. DNA Foot printing
- 43. SHORT ANSWER QUESTIONS 1.Clinical Application of RFLP 2. DNA Foot printing