Blue Illustrated Laboratory Presentation.pptx

Blue Illustrated Laboratory Presentation.pptx

1. MOLECULAR DETECTION OF DISEASE DUE TO GENETIC MUTATION AIZA MALIK 2021-MLTE-001 HIRA RIAZ 2021-MLTE-002

2. INTRODUCTION Genetic mutations refer to alterations in the DNA sequence which affects the amount of gene product produced. Molecular diagnostics map health accurately at molecular level and detect biological molecules (biomarkers) associated with genetic diseases in patient samples. Biomarkers can be specific proteins, DNA or RNA sequences, or metabolites that are present (or present at altered levels) in body fluid or tissue samples of affected individuals compared to healthy individuals.

3. To detect and determine a particular disease To identify cause of disease To predict the likelihood of developing a disease To monitor the progress of a disease. OBJECTIVE As these DNA-based approaches can diagnose the disease at the genetic level, so they are now widely used in personalized medicine for various purposes such as

4. MOLECULAR DIAGNOSTIC TECHNIQUES Molecular diagnostics for genetic mutations involve various techniques aimed at identifying specific mutations or variations in the DNA sequence associated with diseases or conditions. Since there is no single perfect method to screen for unknown mutations so combinations of these methods may be necessary for accurate genetic diagnosis.

5. Some commonly used molecular diagnostic methods for genetic mutations are Polymerase Chain reaction (PCR) Multiplex Ligation-dependent Probe Amplification (MLPA) High-Resolution Melting Analysis (HRMA) Fluorescence In Situ Hybridization (FISH) DNA microarrays Sequencing Restriction Fragment Length polymorphism (RFLP) Southern Blotting

6. Polymerase Chain reaction (PCR) The applications of polymerase chain reaction (PR) technology to genomic screening have made rapid and accurate genetical diagnosis possible.It is a widely used molecular biology technique involves repeated cycles of DNA denaturation, primer annealing, and DNA extension using a heat-stable DNA polymerase enzyme.It amplifies specific regions of DNA, allowing for the detection of mutations in genes of interest.

7. Following PCR techniques are used to identify specific mutations associated with various genetic disorders. real-time PCR allele-specific PCR digital PCR multiplex PCR

8. Real time PCR also known as quantitative PCR allows for the quantification of DNA during the amplification process in real-time. It uses fluorescent probes or DNA-binding dyes to measure the accumulation of amplified DNA with each cycle of PCR. qPCR is highly sensitive and accurate, making it suitable for detection of genetic mutations associated with diseases Real time PCR

9. Allele-specific PCR (AS-PCR) is a modification of standard PCR that amplifies only the allele containing the mutation of interest. This technique uses allele-specific primers designed to specifically amplify the wild-type or mutant allele. AS-PCR is particularly useful for the detection of specific genetic variations associated with inherited diseases or polymorphisms. Allele-specific PCR

10. dPCR is a highly sensitive method for quantifying nucleic acids. It can be used to detect and quantify rare mutations present at low levels in a sample. dPCR is extensively used in detecting mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Limitations necessity of highly allele-specific probes a large sample volume. Digital PCR

11. Multiplex PCR allows for the simultaneous amplification of multiple target sequences in a single reaction. It uses multiple primer pairs targeting different regions of the genome, enabling the detection of multiple mutations or targets in a single PCR assay. Multiplex PCR is useful for its application in genetic screening Multiplex PCR

12. Multiplex Ligation-dependent Probe Amplification (MLPA) is used to detect deletions, duplications, and other copy number variations in specific genes.It can be particularly useful for identifying mutations associated with disorders caused by structural variations in the genome.MLPA assay can also be used in the molecular diagnosis of genetic diseases characterized by the presence of abnormal DNA methylation. Due to the large number of genes that can be analyzed by a single technique, MLPA assay represents the gold standard for molecular analysis of all pathologies derived from the presence of gene copy number variation. MLPA

13. High-Resolution Melting Analysis (HRMA) is a molecular diagnostic technique used to detect genetic mutations by analyzing the melting behavior of PCR-amplified DNA fragments. It begins with the amplification of the target DNA region using PCR, followed by a gradual increase in temperature to denature the double-stranded DNA into single strands. As the DNA strands separate, the fluorescence of a DNA-binding dye, such as SYBR Green, decreases, generating a melting curve that reflects the melting behavior of the amplified DNA fragments. Mutations within the target DNA region alter the melting characteristics, resulting in a shift in the shape or position of the melting curve compared to the wild-type sequence. HRMA

14. FISH is used to detect chromosomal abnormalities or gene rearrangements associated with certain genetic disorders. It involves the hybridization of fluorescently labeled probes to specific DNA sequences on chromosomes. FISH

15. In molecular diagnostics, sequencing is used to identify genetic mutations by determining the precise order of nucleotides in a DNA sample. This can involve techniques like Sanger sequencing Next-generation sequencing (NGS) These can detect various types of mutations such as single nucleotide polymorphisms (SNPs), insertions, deletions, and rearrangements. The sequence data is then analyzed to pinpoint mutations associated with diseases or genetic conditions. Sequencing

16. Sanger sequencing is a traditional method for determining the nucleotide sequence of DNA. It can be used to directly sequence genes and identify mutations within them. Sanger Sequencing

17. NGS technologies enable the rapid and cost-effective sequencing of large portions of the genome or specific genes. Whole exome sequencing (WES) and targeted gene panel sequencing are used to identify mutations associated with inherited diseases. NGS

18. DNA microarrays in molecular diagnostics utilize probes immobilized on a solid surface to simultaneously analyze thousands of genes or genetic variations. Patient DNA, labeled with a fluorescent dye, is hybridized to these probes. If the patient's DNA contains mutations or variations complementary to the probes, they bind to the corresponding probes on the microarray. Through scanning the microarray, fluorescence signals indicate successful hybridization. Data analysis of the intensity and patterns of fluorescence helps identify genetic mutations or variations present in the patient's sample. DNA microarray

19. RFLP (Restriction Fragment Length Polymorphism) is a technique used to detect genetic variations, including mutations. It involves analyzing DNA fragments that result from digesting a DNA sample with specific restriction enzymes. By comparing the lengths of these fragments between individuals, researchers can identify variations such as mutations associated with diseases. RFLP has been particularly useful in identifying mutations linked to genetic disorders like sickle cell anemia and cystic fibrosis. RFLP

20. Applications Disease study Parental testing Criminal suspects Advantage Tells either individual is homozygous or heterozygous Disadvantage Costly

21. Southern blotting is a molecular technique used to detect specific DNA sequences, including genetic mutations. It involves fragmenting DNA samples, separating the fragments by size via gel electrophoresis, transferring them onto a membrane, and hybridizing them with labeled probes complementary to the target sequence. The presence of the target sequence is then detected through probe binding. In mutation detection, Southern blotting enables the identification of mutations by comparing the presence or absence of specific DNA sequences between individuals or between healthy and affected samples. Southern Blotting

22. Cystic Fibrosis: Autosmal recessive disorder Due to abnormality in function and production of CFTR(Transmembrane conductance regulator) Methods of detection: 1. Hybridization Genetic diseases PCR HYBRIDIZATION DETECTION BLOTTING

23. Genetic Diseases

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