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DNA analysis
This document discusses various techniques used in DNA analysis, including blotting techniques like Southern blot, Northern blot, and Western blot. It describes restriction fragment length polymorphism (RFLP) and how it can be used to detect genetic defects. It also explains polymerase chain reaction (PCR), a technique that amplifies specific DNA sequences, allowing them to be analyzed. PCR involves denaturing DNA, annealing primers, and extending new strands in repeated cycles. Applications of PCR include clinical diagnosis, DNA sequencing, and forensic medicine. Overall, the document provides an overview of key analytical techniques used to study DNA, such as blotting, RFLP, and PCR.
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DNA analysis
- 1. DNA analysisDNA analysis Prof.Dr. V. P. AcharyaProf. Dr. V. P. Acharya MBBS, MD, PhDMBBS, MD, PhD
- 2. • Can takemany approaches from simple quantification to complete sequence • Common forms of analysis are – size of DNA - electrophoresis – homology of DNA - blotting, PCR – arrangement of DNA - blotting, PCR, sequencing
- 3. Blot transfer techniquesBlottransfer techniques • Southern blot– DNA • Northern blot– RNA • Western blot– Protein • Technique to visualise specific DNA, RNA and Protein among other molecules
- 4. Southern BlotSouthern Blot •E. M. Southern– 1976 • Useful for quantitating a specific gene • Know mutation in the gene
- 5. • DNA extracted •Digested with restriction endonucleases • Fragments separated by gel electrophoresis • After a suitable time, DNA denatured by mild alkali to ssDNA • DNA transferred to nitrocellulose membrane (Blot) • Paper exposed to cDNA probes • Paper exposed to X-ray– specific bands seen
- 6. Application of SouthernblotApplication of Southern blot • Mutant genes detected e.g. HbS, Cystic fibrosis, PKU, Viral DNA • Forensic purpose • RFLP for pathological disease • Confirmation of DNA cloning results
- 7. Northern BlotNorthern Blot •Size and quantitate specific RNA molecule • Alkali not used • cDNA probes are used- RNA-DNA Hybridisation • Similar to Southern blot Application: Detection of gene expression in a tissue
- 8. Western BlotWestern Blot •Size and quantitate specific protein molecule • No complementary substance required Application: Detection of HIV viral proteins- confirmatory test
- 9. Dot blot/ Slotblot techniqueDot blot/ Slot blot technique • Modified Western blot technique • No electrophoresis- sample directly applied as a dot on the blot paper and analysed • Can quantify DNA, RNA or specific proteins
- 10. Restriction Fragment lengthPolymorphismRestriction Fragment length Polymorphism • Variation in DNA sequence– Polymorphism • Occurs once every 500 nucleotides &107 times per genome • Normally takes place in non-coding regions • These variations cause variation in the restriction sites and thus length of restriction fragments • Inherited difference in the pattern of restriction sites- RFLP
- 11. Application of RFLPApplicationof RFLP • Detects genetic defects in parents and fetal tissues e.g. sickle cell anaemia, Retinoblastoma, Alzeihmer’s disease • Forensic medicine– DNA fingerprinting
- 12. VNTR (Variable numbertandem repeats)VNTR (Variable number tandem repeats) • Unique to each one • Minisatellites • Different numbers of base sequences between 2 points of DNA • DNA fingerprinting VNTR (D1S80) in 6 individuals
- 13. Microsatellites/ Simple tandemMicrosatellites/Simple tandem repeats (STR)repeats (STR) • 2-3 nucleotides • Evenly distributed throughout the genome • More preferred over VNTR • Can be identified by PCR
- 14. DNA SequencingDNA Sequencing •Maxam Gilbert technique • Sanger’s method- interrupted enzymatic cleavage • Automated DNA sequencing • DNA chips- Microarray
- 15.
- 16. Polymerase Chain reaCtionPolymeraseChain reaCtion • Karry Mullis– 1994 • Works like a photocopier • PCR is a technique that takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing.
- 17. PCR targetsPCR targets Thetargets in PCR are the sequences of DNA on each end of the region of interest, which can be a complete gene or small sequence. The number of bases in the targets can vary. TTAAGGCTCGA . . . . AATTGGTTAA The . . . . Represents the middle DNA sequence, and does not have to be known to replicate it.
- 18. Requisites for PCRRequisitesfor PCR • DNA nucleotides, the building blocks for the new DNA • Template DNA, the DNA sequence that you want to amplify • Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA • DNA polymerase (Taq Polymerase, Tth polymerase), a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA
- 19. Steps of PCRStepsof PCR • Denaturation—940 c • Annealing—540 c • Extension-- 72°C • 20-40 cycles, Thermocycler
- 21. Exponential increase inthe no. of copies of DNAExponential increase in the no. of copies of DNA
- 22. 3 stages ineach cycle of PCR3 stages in each cycle of PCR
- 23. It can’t goon infinitelyIt can’t go on infinitely • Saturation • Competition for primers • Rapid cycling- less non- specific products • Size matters- primer length ideal between 18- 25bp • Small length DNA should be chosen to be copied
- 24. Variations of PCRVariationsof PCR • RT-PCR • Nested PCR • Real-time PCR • Multiplex PCR
- 25. Applications of PCRApplicationsof PCR • Clinical diagnosis Prenatal diagnosis Retroviral infections Bacterial infections cancers • DNA sequencing • Comparative studies of genome • Forensic medicine
- 26. Other forms oftarget amplificationsOther forms of target amplifications • Transcription-based amplification method • Strand displacement amplification • Loop-mediated amplification • Whole genome and whole transcriptome amplification
- 27.
Editor's Notes
- #19 Tth- capability to act on both DNA polymerase and reverse transcription activities