& ‘Spectrum of Innovation CHROMOUS BIOTECH PV. RESTRICTION FRAGMENT LENGTH POLYMORPHISM TEACHING KIT INTRODUCTION: * Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms ‘may be differentiated by analysis of pattems derived from cleavage of their DNA + Iftwo organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the pattems generated can be used w differentiate species (and even strains) from one another. * Restriction fragment length polymorphisms (RFLPs) were the first type of molecular ‘markers used in linkage studies * RELPs arise because mutations can create cr destroy the sites recognized by specific yestriction enzymes, leading to variations between individuals in the length of restriction fragments produced from identical regions of he genome * Differences in the sizes of restriction fragments between individuals can be detected by Souther blotting with a probe specific for a segion of DNA known to contain an RFLP The segregation and meiotic recombin: 1 of such DNA polymorphisms can be followed like typical genetic markers. RFP analysis of a family can detect. the Segregation of an RFLP that can be used to tst for statistically significant linkage to the allele for an inherited disease or some other ht man trait of interest. * RFLP markers have several advantages in -omparison with the RAPD and isozyme markers: 1) They are codominant and unaffected by the environment: 2). Any source DNA can be used for the analysis: 3) Many markers can be mapped in a populat on that is no‘ stressed by the effects of phenotypic mutations. ¢ RELPs have provided valuable information in nany areas of biology. including * screening human DNA for the presence of potintially deleterious genes * Providing evidence to establish the innocence vf, or a probability of the guilt of, a crime suspect by DNA fingerprinting + RFLPis suited for construction of linkage mars | “Typieal RELP profiles MTK30 Page | of 5‘Spectrum of Innovation CHROMOUS BIOTECH PVT. LTD, Same segment of UA em tet in aus: _ Desrammate epeenation of Keston kg pomorphism ehmique KEP DESCRIPTION: the kit is designed to simulate the RFLP methodology unknown sample are provided with kit, RFLP ie perl unknown sample with that of the rele Mogments are viewed under UV light PRECAUTION {she insteucton ‘anual before starting the experiment } * the staining dye contains Ethidium Bromide this is known to be carcinogenic and hence k 'o ik be handled with extreme care, Use wloves while handling the staining dye and the , \wi,r0se vel. i * Enzymes are sensitive to temperature Always Keep the enzymes at -20°C. 3 Switch on the dry bath, before starting atid set the temperature to 37°C * Prepare 196 auarose vel before setting up th restriction digestion reaction. All the sarose gel (0 sel For Ihr at room temperature ° The reagents provided for preparing and runnin A rose gels are enough for casting and sat 3 Res: of #6 gels OF 30m volume exch, Minimure tee periments are to be ‘“efout simultaneously and samples ave loaded ont te same cer MIk30 Page 2 of 5g Spectrum of Innovation CHROMOUS BIOTECH PVT, :10, KIT COMPONENTS: The components should be stored as suggested for best results, Use the kit within lyear of arrival. The kit contains the following components, Materials provided are sufficient for 5 experiments. » [Component ~ | Quantity Storage Restriction enzyme (Hindi) 30.ul 20°C 10 X Assay buffer 60-41 Ca Unknown sample 75 al -20°C Reference sample 1 75 al 20°C} Reference sample 2 75 jal -20°C Reference sample 3 Tkb DNA Ladder (Ready-to-Toad) "| Agarose 6X GLB 50X TAE Staining dye 1.5 ml vials _ Water LO ol -20°C MATERIALS REQUIRED (Not included in the 1 it): Dry bath, crushed ice, tips, micropipette ete. PROCEDURE: Note: Minimum two experiments should be ¢ id out simultaneously, Setting up of Restriction digestion reaction: * Three reference samples and one unknown sample is digested with Hind II restriction enzyme, * Four separate reactions has to be set up. Remove the Reference samples, unknown DNA samples; assay buffer kept in -20°C and ut them on crushed ice. Allow the components to get thawed on ive * Prepare the reaction mix as given below. Ad all the components in the same order as, given below: Hind TIT reaction mixture: DNA sample 15.0 pl 10 X Assay buffer 3.0 ul Hind Ill enzyme (SU/pl) 15 ul Water 10.5 ul Total 30.0 ul * Add all the components for the mix and tap it 2-3 times for mixing. Give a shor spin at 10000 rpm for 20 sec MTk30 Page 3 of 5Spectrum of Innovation Agarose gel electrophoresis: Fig: 1% agarose gel showing restriction profiles of Reference samples and profile of unknown sample loaded along with 1kb DNA ladder we Mrk30 (CHROMOUS BIOTECH PVT. LTD. * Incubate the mix at 37°C dry bath for | hour Prepare | % Agarose gel before setting up the restriction digestion reaction, Weigh 05g of agarose and add 50 ml of 1X TAE. Boil the mix to melt the agarose completely. Pour the gel into appropriate gel casting assembly. Add 200 ml 1X TAE to the gel and add 5 ul of staining dye to the buffer and mix by tilting the gel tank 5-6 times. Pre run the gel for 5 min before loading the samples. After the restriction digestion reaction, add 3 lof gel loading dye to each of the reaction. mix in vials L-oad the entire volume of digested samples on the Agarose gel. Load 20 il of 1 kb ladder (ready-to-load) along with the digested samples. Electrophorese the samples for I hr at 100 V Visualize the gel under UV transilluminator, RVATION: Observe the DNA banding pattem obtained by three different reference samples and one unknown DNA sample. Compare the unknown sample with that of the reference sample. Lane description: Restriction profile of reference samples and unknown samples 1, Reference Sample 1 2. Reference Sample 2 3. Reference Sample 3 4. Unknown Sample 5. Ikb DNA ladder a PRETATION: From the restriction profiles obtained, one can note that restriction profile of unknown sample matches with the reference sample 2. - The restriction enzyme recognizes and cuts only a particular base sequence unique to it. Even a slight change occurring in the nucleotide bases will results in loss of recognition site for a particular restriction enzyme. Hence giving rise to different banding pattem, Page 4 of 5‘Spectrum of Innovation * RFLP technique helps in identifying a particu ar trait of a # few mutations in the restriction enzyme targ st sequence Mrk30 CHROMOUS BIOTECH PVT. LID. in organism that can be linked 10