Molecular Genetics & Genetic Disorders PDF

MOLECULAR GENETICS & GENETIC DISORDERS NORLELAWATI BINTI A TALIB 2425 LECTURES OBJECTIVES o Explain the molecular basis of genetics, including the structure, function, and expression of genes. o Describe different types of mutations and their consequences on human health...

MOLECULAR GENETICS & GENETIC DISORDERS NORLELAWATI BINTI A TALIB 2425 LECTURES OBJECTIVES o Explain the molecular basis of genetics, including the structure, function, and expression of genes. o Describe different types of mutations and their consequences on human health. o Introduce diagnostic techniques and molecular tools used in studying genetic disorders o Discuss the classification and examples of genetic disorders, including their molecular pathogenesis and inheritance patterns. ACTIVITIES Foundation of Molecular Genetics Molecular Diagnostics and Emerging Tools Genetic Disorders -Chromosomal Disorders Genetic Disorders --Mendelian inheritance etc BASIC FOUNDATION IN MOLECULAR GENETICS 1. Introduction to Genetics- terminology 2. DNA structure 3. Genome and Genes 4. Central dogma and gene expression 5. Coding & Non-coding regions 6. Mutation Overview 7. Epigenetics concept KEY GENETIC TERMS & DEFINITION o Genome: Complete set of genes. o Gene: Unit of DNA responsible for protein synthesis. o Locus: Specific site of a gene on a chromosome. o Allele: Variant forms of a gene. o Genotype & Phenotype: Genetic composition vs. observable traits. o Karyotype: Visual representation of chromosome DEFINE THESE GENETIC TERMS 1.Polymorphism: 2.Haploid and Diploid: 3.Epigenetics: 4.Penetrance: 5.Expressivity: 6.Noncoding DNA: 7.Silent Mutation: 8.Frameshift Mutation:. 9.Trinucleotide Repeat Expansion: 10.Gene Heterogeneity: 11.Pleiotropy: 12.Anticipation: 13.Mosaicism: 14.Linkage Disequilibrium: 15.Gene Therapy: STRUCTURE OF DNA Double Helix: Two strands wound around each other.(twisted ladder) DNA is Made of Nucleotides: Base: Adenine (A), Thymine (T), Cytosine (C), Guanine (G). Base Pairing Rules: A-T and C-G​​. (Sugar (deoxyribose), Phosphate, Base) Organization Of DNA Heterochromatin: Euchromatin: Loosely packed, Tightly packed, inactive active for gene expression. regions. DNA wrap around protein long Coiling of nucleosome: chromatin. During cell division, chromatin folds even tighter to form chromosomes; 46 Chr (23 pairs)  CENTRAL DOGMA OF MOLECULAR BIOLOGY FLOW OF GENETIC INFORMATION 1. DNA → RNA → Protein 1. Transcription: DNA To RNA. 2. Translation: RNA To Protein. 2. Enzymes Involved: RNA Polymerase 3. Ribosomes. TRANSCRIPTION -RNA SPLICING-TRANSLATION Process of Transcription DNA is unwound by RNA polymerase. mRNA is synthesized using the DNA template strand. mRNA sequence: Complementary to DNA (A pairs with U in RNA). RNA Processing Removal of introns (non-coding regions). Joining of exons (coding regions). Formation of mature mRNA for translation. Protein Synthesis mRNA is read by ribosomes. tRNA brings amino acids corresponding to codons. Start Codon: AUG (Methionine). Stop Codons: UAA, UAG, UGA.  GENETIC CODE Features of the Genetic Code Triplet code: 3 nucleotides = 1 codon. Universal and degenerate (multiple codons for one amino acid). Examples: AUG = Methionine (Start). UAA/UAG/UGA = Stop codons.  GENOME’S “DARK MATTER”= NONCODING REGIONS o Long-stretches DNA o Mainly devoted to regulation of gene expression. o “architectural planning” vs “construction materials”. o LCR (locus control region) o eg. LCR for HBB genes CODING & NON-CODING RNA  INTRODUCTION TO MUTATION o Permanent alterations in the DNA sequence o Inherited or acquired o Although DNA replication is a very accurate process, mistakes can occur producing changes. o Radiation, Chemical, UV light. o Can occur in coding (exons) or non-coding (introns, regulatory regions,splicing) regions o Basis of genetic variability in some diseases CATEGORIES OF MUTATION Genome mutation scale Chromosome mutation Gene mutation location Germline mutation Somatic mutation Silence mutation Missense mutation effects Nonsense mutation Loss of function mutation Gain of function mutation Where is the location of the mutation? G e r m L i n e V s S o m a t i c M u t a t i o n How much is the DNA’s changed and lost? loss or gain of whole chromosomes Genome mutation monosomy, trisomy Chromosomal rearranged genetic material - visible changes mutation in chromosome structure. submicroscopic mutation. Partial or complete deletion of a gene or often a single base change Gene mutation Point mutations ;Frameshift mutations ;Insertion or deletion Splicing mutation; Termination Mutations Trinucleotide repeat mutations  Silent Mutation: No change in protein function. Missense Mutation: Amino acid substitution (e.g., β-Thalassemia). Nonsense Mutation: Premature stop codon. Loss-of-Function Mutation: Reduced/absent protein activity (e.g., Cystic Fibrosis). Gain-of-Function Mutation: Enhanced protein function or new function (e.g., Huntington's Disease).  EPIGENETICS o Heritable Changes In Gene Expression That Do Not Involve Alterations In The DNA Sequence. Key Mechanisms: 1. DNA Methylation: Addition of methyl groups to cytosine, typically silencing genes. 2. Histone Modifications: 1. Acetylation: Opens chromatin, promoting gene expression. 2. Methylation: Can either activate or repress genes. 3. Non-Coding RNAs: Regulate gene expression at transcriptional and post- transcriptional levels.  CLINICAL APPLICATION OF EPIGENETICS Cancer Epigenetics: Drugs Targeting DNA Methylation (eg, Decitabine). Developmental Disorders: Role Of Imprinting In Diseases Like Beckwith-wiedemann Syndrome. miRNA Therapeutics: Target miRNA Implicated In Cancers. RNA Interference (RNAi): siRNA- based Therapies Epigenetic Biomarkers For Early Disease Detection. BASIC FOUNDATION IN MOLECULAR GENETICS 1. Introduction to Method in Diagnosis of Genetic Disease. 2. Concept of karyotyping vs molecular test 3. Introduction on molecular applications of genetic test 4. PCR basic concept & application 5. NGS basic concept & application Method in the diagnosis of genetic disorders o Genetic test is required for Disorders Requiring Genetic Characterization: o To Confirm Diagnosis,. o Guide Treatment o Provide Genetic Counseling o Cytogenetic Analysis (Large Chromosomal Abnormalities) o Molecular Test (Precise Genetic Changes).  Methods For Mutation Analysis Detects large PCR: Amplifies DNA to detect small chromosomal mutations eg changes (e.g., GAP-PCR for Alpha Thal.. trisomies, translocations). DNA/RNA Cytogenetic & Chromosome Sanger sequencing /karyotyping Pyrosequencing Southern blot FISH Next generation PCR sequencing(NGS) qRT-PCR Digital PCR DNA/RNA CHROMOSOME &Chromosome High-throughput sequencing for gene panels or whole exome/genome. Eg FISH:Identifies specific DNA BRCA sequencing sequences and structural abnormalities.eg: BCR-ABL1 in CML Karyotyping / Cytogenetic Analysis  Methods For DNA Analysis (past-present-future) Epigenetic Testing: Assesses DNA methylation patterns and histone modifications. Traditional Southern Blotting PCR and Sanger Advanced method Laborious and time Sequencing NGS consuming NGS: enabling high-throughput analysis of multiple genes simultaneously.  PRINCIPLE OF POLYMERASE CHAIN REACTION Tools o Some basic concepts o DNA is denatured at high Cycling of temperature temperature o DNA can be reannealed back at Good lower temperature Recipe enzymes o DNA can be synthesized o Specific DNA sequence can be copied Sequence and target o amplified primers  e.g Hotstar Taq Polymerase e.g Primers from ITDNA Thermocycler Basis of Polymerase Chain Reaction (PCR) Conventional PCR products on agarose gel Conventional Vs Real Time PCR  REAL-TIME PCR ( QRT-PCR) Quantitative real time PCR (qRT-PCR) Gene expression Viral loads Copy number analysis Genotyping analysis Diagnostic application Detection of pathogen Mutation Detection Etc. Evolution of DNA sequencing techniques Sanger sequencing / Direct nucleotide sequencing o Parallel sequencing of multiple small fragments of DNA. o High through put, speed and scalability  Next Generation Sequencing (NGS) o Parallel sequencing of multiple small fragments of DNA. o High through put, speed and scalability THANK YOU NORLELAWATI BINT A TALIB