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Services related to Genetics _ Genetic Testing.pptx
- 1. Services related to Genetics : Genetic Testing Dr. Atanu Kumar Dutta Additional Professor Department of Biochemistry AIIMS Kalyani B.Sc. Nursing
- 2. Definition • Genomics is defined as the study of genes and their functions, and related techniques (WHO). • Human Genome: The human genome is the complete set of nucleic acid sequence for humans (Homo sapiens). Human genomes include both protein-coding DNA genes and noncoding DNA. • 3,234.83 Mb (Mega-base pairs) per haploid genome. • Human Genome Project: First draft February 12, 2001.
- 3. What is genetic testing? • Genetic testing is a type of medical test that identifies changes in genes and chromosomes 3
- 4. What are the different types of genetic tests? • Targeted single variant • Single gene • Gene panel • Whole exome sequencing/whole genome sequencing 4
- 5. Targeted mutation analysis-Single mutation • Disease shows genetic homogeneity at variant level • One or few variants known to be responsible for majority of patients • Definitive clinical features or diagnostic tests • Examples • Sickle cell anemia--- NM_000518.4(HBB):c.20A>T,p.Glu6Val • Achondroplasia --- NM_000142(FGFR3):c.1138G>A • Apert syndrome--- NM_000141.4(FGFR2):c.755C>G 5
- 6. Targeted mutation analysis-Single mutation 6
- 7. Amniocentesis β globin gene Targeted mutation analysis - Small gene Beta thalassemia
- 8. Next Generation Sequencing Refers to all post-Sanger sequencing technologies that enable massive sequencing at low cost 8 Massive: Millions of DNA fragments are sequenced Parallel: All fragments are sequenced simultaneously Deep: Each fragment is sequenced a number of times Resequencing: Generated sequence is aligned to a reference genome
- 9. Exome sequencing (WES): Coverage of reads restricted to exons of genes Clinical Exome: covers exons of nearly 7000 genes with reported OMIM phenotype Disease specific panels: usually 50-200 genes specific to the phenotype like deafness, retinitis pigmentosa, hereditary cancer syndromes, etc Whole genome sequencing (WGS): covers the entire genome Trio-Sequencing: Proband + Parents 9 Image : N Engl J Med 2018;379:1353-62. Clinical NGS
- 10. What are the uses of genetic testing? • Newborn screening • Diagnostic testing • Carrier testing • Prenatal testing • Preimplantation testing • Predictive and presymptomatic testing • Forensic testing 10
- 11. How is genetic testing done? 11
- 12. Polymerase chain reaction - PCR 12 PCR-RFLP, qPCR, DNA Sequencing, etc
- 13. Quantitative PCR As the name suggests used to estimate the amount of nucleic acid material in the sample • End Point PCR • Signal from the fluorescent probes is measured at the end of PCR reaction • Real time PCR • Signal from the fluorescent probes is measured during the PCR reaction 13
- 14. End Point PCR 14 Fluorescent signal is measured after the end of PCR – makes it less sensitive and inaccurate. (Semiquantitative) Modifications in the techniques (eg- Multiplex ligation-dependent probe amplification) – to ensure accuracy Eg- Aneuploidy testing by QF-PCR, MLPA QF-PCR MLPA
- 15. Real time PCR Fluorescent signal is plotted against PCR cycles Cycle Threshold (ct) - PCR cycle number where the fluorescence from the reporter is greater than threshold level It is inversely proportional to the initial quantity of DNA/RNA Used for • Quantitative analysis of gene expression, viral loads • CNV validation • targeted SNP analysis 15 • Sensitive & Accurate • Rapid • Requires specific equipment and slightly costlier E-gene amplification in a Covid-19 infected individual
- 16. Sanger sequencing 16 DNA PCR with specific primer PCR product Single primer PCR with dideoxy nucleotides Chromatogram Capillary eletrophoresis
- 17. Sanger sequencing 17 Homozygous variation Heterozygous variation Heterozygous insertion/deletion Normal
- 19. What is informed consent? • Example 19
- 20. How can I be sure a genetic test is valid and useful? • Analytical validity • Clinical validity • Clinical utility 20
- 21. What do the results of genetic tests mean? 21
- 22. What are the benefits of genetic testing? • Early intervention • Reducing diagnostic time • Family screening • Prenatal testing • Targeted therapy 22
- 23. What are the risks and limitations of genetic testing? • Medical • Emotional • Social • Financial • Discrimination 23
- 24. Thank You 24
Editor's Notes
- Picture shows evolution of technologies over the years….
- The human genome comprises of 3.2 billions (GB) base pairs organised in 22 autosomes and two sex chromosomes. Only ~ 2% of this sequence is actually coding, and results in downstream processing with protein formation. The function of large protions of remaining intergenic regions and non-coding intragenic regions remains unclear. Within a gene, coding regions are called Exons and non-coding regions are called introns. It is expected that the cause of a genetic disorder is more likely to be due to a variation in coding regions, so to save time and money, these regions termed ‘Exome’ can be sequenced for diagnosis in majority of cases. Trio-exome is especially helpful in denovo variants WGS in addition to covering the entire genome for single nucleotide variants is good for detection of copy number variants and structural variants which makes it a very comprehensive but expensive test
- Method developed by Kary Mullis in 1980s Ability of a thermostable DNA polymerase to synthesise complementary strand is exploited in this technique. Each cycle consists of Denaturation – separation of DNA strands Annealing – Primers getting attached to the template Extension – synthesis of complimentary strand Repetition of the above steps results in exponential increase in PCR products
- QF PCR- quatitative flouresence PCR – Used for rapid detection of aneuploidies in prenatal samples MLPA – DMD and SMA common use case scenarios….good for carrier detection. Image shows deletion of exon 51 and 52, ratios of test samples with references are used for interpretation….. 0 indicates absence/homozygous deletion of the target region; values around 0.5 for heterozygous deletion or carrier status; 1- normal; 1.5 and above duplications
- Exponential phase of the PCR is measured for quantitation in contrast to end-point where the plateu phase is measured Higher amount of template DNA results in requirement of lesser number of cycles for the signal to be detected, thus inversely proportional Different types – based on DNA binding dye/ fluorescent probes/primers available….latter being more specific and accurate
- Random incorporation of dideoxy nucleotides generates fragments of different length which are fluorescently labelled according to the last nucleotide incorporated (ddNTP). The fragments are separated by capillary electrophoresis Laser excitation is used to identify the flourophore and thus the nucleic acid. Data is integrated and presented as a chromatogram by computer software
- Heterozygous variation – two colored peaks at the same position Homozygous variation – Single peak with an alternate colour Heterozygous ins/deletion – two peaks continuously from a point in otherwise a clean sequence
- The DNA is broken down in to small fragments….further processing called library preparation prepares these fragments for sequencing. Sequencing in most clinical applications happens when a complimentary strand is being synthesized. Signals are captured and recognized when a new base gets incorporated. Sequence from each individual fragment (read) are now aligned to a reference genome and variants are detected based on the mismatches These variants get annotated and undergo further down stream analysis for identification of the causative variation