logging in or signing up Molecular Diagnostic methods raniashok Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 2237 Category: Education License: All Rights Reserved Like it (6) Dislike it (0) Added: November 17, 2009 This Presentation is Public Favorites: 1 Presentation Description Deals with molecular screening techniques, PCR etc. & its applications Comments Posting comment... By: maxefui (12 month(s) ago) Good images. it will work better if you add presentation notes Saving..... Post Reply Close Saving..... Edit Comment Close By: prof_mibi (13 month(s) ago) excellent presentation! could you pls send it to hesty_l@yahoo.com.Thanks. Saving..... 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See all Premium member Presentation Transcript Slide 1: Rani Ashok, Lecturer (SS) in Zoology, Lady Doak College, Madurai - 2 MOLECULAR DIAGNOSTIC METHODS Blotting methods : Blotting methods Radioactive Probe Hybridization : Radioactive Probe Hybridization Probes complementary to the sequence of interest can be used to detect the amount and location of the desired sequence present in the gel Analytical Blots : Analytical Blots Southern Northern Western DNA mRNA Protein Detects: Probe: Nucleic acid Nucleic acid Antibody gel blot gel blot gel blot Slide 9: SOUTHERN BLOTTING Restriction Enzyme Mapping of Fragments : Restriction Enzyme Mapping of Fragments Digestion with enzyme 1 shows that there are two restriction sites for this enzyme but does not reveal whether the 3kb fragment is in the middle or on the end. Digestion with enzyme 2 shows that the 17kb fragment has a single site. The double digest shows that the 8kb and 6kb fragments from enzyme one are retained and that the 3kb fragment is cut by enzyme 2. Thus the 3kb fragment is in the middle. The double digest fragments indicate that the 6kb fragment plus the 1kb fragment make up the 7kb fragment identified by enzyme 2, while the 8kb + 2kb fragments make up the 10kb fragment identified by enzyme 2. Expression profiling using microarrays : Expression profiling using microarrays Slide 14: PCR REQUIREMENTS for PCR Two primers · flank region you are interested in · you must know the sequence of the flanking regions so you can order appropriate primers Heat stable polymerase Four dNTPs Thermocycler (standard, but optional) · changes temperature very rapidly for each cycle (denature, anneal, extend) Slide 15: Programmable thermocycler Slide 16: PCR METHOD There are four basic steps in PCR 1. Denaturing Stage 2. Annealing Stage 3. Extending Stage 4. Replication Slide 17: PCR METHOD – DENATURING STAGE Slide 18: PCR METHOD – ANNEALING STAGE Slide 19: PCR METHOD – EXTENDING STAGE Slide 21: PCR METHOD - REPLICATION Each cycle doubles the copies of double stranded DNA. DNA Cycle 1 2 3 4 5 … The average number of cycles performed with PCR, for efficiency reasons, is thirty. Amplified Fragment Length Polymorphism : Amplified Fragment Length Polymorphism Polymorphism based on gain or loss of restriction site, or selective bases Technically demanding and expensive Many markers generated, mostly dominant More reliable than RAPD, less so than SSR No prior sequence knowledge required Single-Strand Conformational Polymorphism : Single-Strand Conformational Polymorphism Highly sensitive to DNA sequence: can detect single base changes Simple process but can be difficult to repeat 1. Amplify Target Sequence 2. Denature product with heat and formamide 3. Analyze on native (nondenaturing) polyacrylamide gel 4. Base sequence determines 3-dimensional conformation, and rate of migration Denaturing Gradient Gel Electrophoresis : Denaturing Gradient Gel Electrophoresis 4. Denaturing gradient gels are difficult to produce: use perpendicular gradient to identify optimal conditions, move to CDGE: constant denaturant gel electrophoresis 1. Amplify Target Sequence 2. Run product on gel with denaturing gradient (parallel or perpendicular to direction gel runs) 3. Product begins denaturing at a certain point, depending on base sequence: greatly retards migration and allows discrimination of alleles based on small sequence differences Cleaved Amplified Polymorphic Sequence : Cleaved Amplified Polymorphic Sequence Fairly simple analysis (cutting can be a hassle) Requires sequence information from several alleles (or luck) 1. Amplify Target Sequence 2. Cut with a restriction enzyme that differentiates alleles 3. Alleles can be differentiated by size based on loss or gain of restriction site; May be able to analyze on agarose gel X Allele 1 Allele 2 Spectrofluorometric Thermal Cycler for Allele Discrimination & Quantitative PCR : Spectrofluorometric Thermal Cycler for Allele Discrimination & Quantitative PCR CSL has an ABI PRISM 7700 Sequence Detection System Real-time detection: Each cycle produces a fluorescent signal proportional to the amount of PCR product present Allele Discrimination : Allele Discrimination Allele Discrimination Assay Overview : Allele Discrimination Assay Overview You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
Molecular Diagnostic methods raniashok Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 2237 Category: Education License: All Rights Reserved Like it (6) Dislike it (0) Added: November 17, 2009 This Presentation is Public Favorites: 1 Presentation Description Deals with molecular screening techniques, PCR etc. & its applications Comments Posting comment... By: maxefui (12 month(s) ago) Good images. it will work better if you add presentation notes Saving..... Post Reply Close Saving..... Edit Comment Close By: prof_mibi (13 month(s) ago) excellent presentation! could you pls send it to hesty_l@yahoo.com.Thanks. Saving..... Post Reply Close Saving..... Edit Comment Close By: sokkappan (16 month(s) ago) nice and crisp presentation mam. I would like to pickup the information and also the images for my project purpose.will u? Saving..... Post Reply Close Saving..... Edit Comment Close By: Evelyn0286 (27 month(s) ago) Dear Rani, Could you please send a file at evtotaan@gmail.com. Thanks. Evelyn Saving..... Post Reply Close Saving..... Edit Comment Close By: anjupragya (28 month(s) ago) Dear Rani These are nice ppt and a good source for educating the student. If you do not mind, can you please send me a file at kumarsanjay66@yahoo.com Thanks.Sanjay Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript Slide 1: Rani Ashok, Lecturer (SS) in Zoology, Lady Doak College, Madurai - 2 MOLECULAR DIAGNOSTIC METHODS Blotting methods : Blotting methods Radioactive Probe Hybridization : Radioactive Probe Hybridization Probes complementary to the sequence of interest can be used to detect the amount and location of the desired sequence present in the gel Analytical Blots : Analytical Blots Southern Northern Western DNA mRNA Protein Detects: Probe: Nucleic acid Nucleic acid Antibody gel blot gel blot gel blot Slide 9: SOUTHERN BLOTTING Restriction Enzyme Mapping of Fragments : Restriction Enzyme Mapping of Fragments Digestion with enzyme 1 shows that there are two restriction sites for this enzyme but does not reveal whether the 3kb fragment is in the middle or on the end. Digestion with enzyme 2 shows that the 17kb fragment has a single site. The double digest shows that the 8kb and 6kb fragments from enzyme one are retained and that the 3kb fragment is cut by enzyme 2. Thus the 3kb fragment is in the middle. The double digest fragments indicate that the 6kb fragment plus the 1kb fragment make up the 7kb fragment identified by enzyme 2, while the 8kb + 2kb fragments make up the 10kb fragment identified by enzyme 2. Expression profiling using microarrays : Expression profiling using microarrays Slide 14: PCR REQUIREMENTS for PCR Two primers · flank region you are interested in · you must know the sequence of the flanking regions so you can order appropriate primers Heat stable polymerase Four dNTPs Thermocycler (standard, but optional) · changes temperature very rapidly for each cycle (denature, anneal, extend) Slide 15: Programmable thermocycler Slide 16: PCR METHOD There are four basic steps in PCR 1. Denaturing Stage 2. Annealing Stage 3. Extending Stage 4. Replication Slide 17: PCR METHOD – DENATURING STAGE Slide 18: PCR METHOD – ANNEALING STAGE Slide 19: PCR METHOD – EXTENDING STAGE Slide 21: PCR METHOD - REPLICATION Each cycle doubles the copies of double stranded DNA. DNA Cycle 1 2 3 4 5 … The average number of cycles performed with PCR, for efficiency reasons, is thirty. Amplified Fragment Length Polymorphism : Amplified Fragment Length Polymorphism Polymorphism based on gain or loss of restriction site, or selective bases Technically demanding and expensive Many markers generated, mostly dominant More reliable than RAPD, less so than SSR No prior sequence knowledge required Single-Strand Conformational Polymorphism : Single-Strand Conformational Polymorphism Highly sensitive to DNA sequence: can detect single base changes Simple process but can be difficult to repeat 1. Amplify Target Sequence 2. Denature product with heat and formamide 3. Analyze on native (nondenaturing) polyacrylamide gel 4. Base sequence determines 3-dimensional conformation, and rate of migration Denaturing Gradient Gel Electrophoresis : Denaturing Gradient Gel Electrophoresis 4. Denaturing gradient gels are difficult to produce: use perpendicular gradient to identify optimal conditions, move to CDGE: constant denaturant gel electrophoresis 1. Amplify Target Sequence 2. Run product on gel with denaturing gradient (parallel or perpendicular to direction gel runs) 3. Product begins denaturing at a certain point, depending on base sequence: greatly retards migration and allows discrimination of alleles based on small sequence differences Cleaved Amplified Polymorphic Sequence : Cleaved Amplified Polymorphic Sequence Fairly simple analysis (cutting can be a hassle) Requires sequence information from several alleles (or luck) 1. Amplify Target Sequence 2. Cut with a restriction enzyme that differentiates alleles 3. Alleles can be differentiated by size based on loss or gain of restriction site; May be able to analyze on agarose gel X Allele 1 Allele 2 Spectrofluorometric Thermal Cycler for Allele Discrimination & Quantitative PCR : Spectrofluorometric Thermal Cycler for Allele Discrimination & Quantitative PCR CSL has an ABI PRISM 7700 Sequence Detection System Real-time detection: Each cycle produces a fluorescent signal proportional to the amount of PCR product present Allele Discrimination : Allele Discrimination Allele Discrimination Assay Overview : Allele Discrimination Assay Overview