Molecular Methods for Diagnosis of Genetic Diseases

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

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Molecular Methods for Diagnosis of Genetic Diseases

  1. 1. Evolving Molecular Methods for Mutation Detection Mohammad Al-Haggar, MD. Professor of Genetics
  2. 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. 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. 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. 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. 6. Restrictionfragment SouthernBlotting
  7. 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. 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. 9. N M N M N M N M Homozygous normal Homozygous mutant Heterozygous No Signal
  10. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 22. 1993
  23. 23. Mohammad Al-Haggar, MD. Professor of Genetics m.alhaggar@yahoo.co.uk

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