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Molecular Methods for Diagnosis of Genetic Diseases
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Molecular Methods for Diagnosis of Genetic Diseases



Basic steps in the evolution of biotechnologic methods in Diagnosis of Genetic diseases

Basic steps in the evolution of biotechnologic methods in Diagnosis of Genetic diseases



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Molecular Methods for Diagnosis of Genetic Diseases Presentation Transcript

  • 1. Evolving Molecular Methods for Mutation Detection Mohammad Al-Haggar, MD. Professor of Genetics
  • 2. Introduction - Mutations  permanent changes in DNA  in either 1. Germ line (gamete)  all cells of an individual, OR 2. Somatic line  only in % body cells. - HGP 2003  basic molecular defect of most genetic diseases  phenotype-genotype correlations. - Since 2003  methods for detection of mutation are evolving:- 1. Old basic tedious and time consuming experiments. 2. Rapid and high throughput in a narrow spaced laboratory.
  • 3. Methods • Some are used together in succession, Others are changing. 1. Southern blotting 2. Restriction fragment length polymorphism (RFLP) 3. PCR/RFLP (size analysis of PCR products) 4. Amplification-refractory mutation system (ARMS) PCR 5. Heteroduplex migration analysis 6. Single- strand conformational polymorphism (SSCP) analysis. 7. Denaturating gradient gel electrophoresis (DGGE) 8. Denaturating high performance liquid chromatography (DHPLC)
  • 4. 9. Conformation-sensitive capillary electrophoresis (CSCE) 10. Oligonucleotide ligation assay (OLA) 11. Real-time PCR 12. DNA microarrays (DNA chips) 13. High-resolution melt curve analysis (HRM) 14. Sequencing.
  • 5. Southern blotting • DNA digestion by restriction enzyme  restriction fragments (RFLP) of different size and molecular weight based on recognition sites. • Separation on agarose gel by electrophoresis. • Denaturation of DNA fragments by soaking gel in an alkaline solution. • Transfer to nylon membrane. • Hybridization  complementary probes (radio-/ biotin labeled). • Radiography.
  • 6. Restrictionfragment SouthernBlotting
  • 7. Amplification (PCR) RFLP • Amplification of DNA stretch by PCR using labeled nucleotides  restriction fragments • Recognition of mutant alleles especially in heterozygous form. • Use of multiple enzymes  multiplex reaction  electrophoresis.
  • 8. Amplification Refractory Mutation System (ARMS) • Amplification of specific allele  used for common mutations e.g. β-thalassemia. • It consists of 2 complementary reactions and utilizes 3 primers  1 constant (complement to template in both reactions), the other 2 primers differ at 3' terminal, 1 for wild and 1 for mutant allele.
  • 9. N M N M N M N M Homozygous normal Homozygous mutant Heterozygous No Signal
  • 10. Single-Strand Conformational Polymorphism (SSCP) • Screening for the presence or absence of mutation in an amplified exon. • Basis: single DNA strand folds to form a complex 3-D structure maintained by intra- molecular bonds. 80% of mutations  variable folding  variable electrophoretic mobility. • Differential mobility between test and control DNA samples  +ve mutation  sequencing.
  • 11. Heteroduplex migration analysis • Differential migration of DNA heteroduplexes compared to homoduplexes (2 mismatched single strands) run on a polyacrylamide gel. • Heteroduplex  PCR products from a suspected carrier are denatured  allowed to reanneal to a normal PCR product • Useful for detection of carriers of X-linked recessive disorders.
  • 12. Denaturating gradient gel electrophoresis (DGGE) • More demanding procedure than SSCP and heteroduplex migration analyses with a higher sensitivity (over 90%) for identifying mutations especially in survey studies. • Basis: normal and mutant sequences will show different band patterns on a gel. The PCR products are run on a special denaturing gradient gel that contains increasing concentrations of a denaturing chemical e.g. urea.
  • 13. Denaturating high performance liquid chromatography (DHPLC) • Similar to DGGE, detects heteroduplexes owing to their abnormal denaturing profiles. • Basis: differential separation of mismatched heteroduplexes which form after the reannealing of normal and mutant DNA strands. PCR products are injected in a column containing an increasing gradient of mobile phase (acetonitrite) required to elute each homo- or heteroduplex. • It is an extremely sensitive method for detecting base substitutions, small deletions and insertions.
  • 14. Conformation-sensitive capillary electrophoresis (CSCE) • It is a faster technique that achieves a higher throughput than DHPLC in detection of the heteroduplexes using fluorescence technology.
  • 15. Oligonucleotide Ligation Assay (OLA) • A pair of oligonucleotides designed to anneal to adjacent sequences within a PCR product  If perfectly hybridized  joined by a DNA ligase. • Oligonucleotides complementary to normal and mutant sequences are differentially labeled and the products are identified by a computer software.
  • 16. High-resolution melt curve analysis (HRM) • Fluorescent dyes intercalates with double strand DNA (during PCR)  PCR products heated  separation of the two strands. • Fluorescence ↓, as DNA strands dissociate  melting profile depends on the PCR product size and sequence. • It is very sensitive. Its detection capability is dependant upon fragment length, sequence, mutation, PCR quality and analytical equipment.
  • 17. Real-time PCR • Quantitative PCR  multiple hardware platforms for real-time PCR, and fast versions that can complete a PCR reaction in less than 30 minutes.
  • 18. DNA microarrays (DNA chips) • Small devices (coated glass microscope slides)  large numbers of different DNA sequences placed on slide using an automated robot. • Gene Chips  different Oligo DNA sequences produced by in situ synthesis  microscopic and submicroscopic VNTR for the whole genome in a single assay. • Major microarray platforms: for genomic DNA profiling; 1. Comparative genomic hybridization (CGH) arrays  use a two-color scheme  infers VNTR in a test sample compared to a reference sample . 2. Genotyping arrays  No control sample; intensity of the hybridization signal α DNA copies  useful for SNPs.
  • 19. Sequencing • Direct determination of DNA sequences using the new generations of automated sequencers. • Basis  Amplification using fluorescent nucleotides  PCR products electrophoresis  fluorescence signal during its moves through a window  converted into an electronic signal  analysis by a computer  a color graph that signify each nucleotide.
  • 20. Nucleotide enumeration • Nowadays  fully automated (hardware platform). • Up to date revision on gene atlas is sometimes necessary (discovery of new exons in a gene  exon re-numbering).
  • 21. Conclusion • Basic molecular techniques are subjected to modifications and optimizations based on: 1. Genetic disease, 2. Mutation type, and 3. Troubleshoots in lab. • Fixed frames of laboratory technique: 1. Amplification, 2. Digestion, 3. Separation (differential migration), 4. Labeling, and 5. Hybridization.
  • 22. 1993
  • 23. Mohammad Al-Haggar, MD. Professor of Genetics m.alhaggar@yahoo.co.uk