Genetics Flipped Lesson 6.PDF

Genetics Flipped Lesson 6 – Molecular Genetics and Sequencing Molecular Genetics Definition - Refers to all diagnostic methods that examine changes of genetic information in extracted DNA or RNA Molecular genetics for diagnosis - 2 main approaches: o Nucleic acid hybridisation – based on complementa...

Genetics Flipped Lesson 6 – Molecular Genetics and Sequencing Molecular Genetics Definition - Refers to all diagnostic methods that examine changes of genetic information in extracted DNA or RNA Molecular genetics for diagnosis - 2 main approaches: o Nucleic acid hybridisation – based on complementarity o Nucleic acid amplification – making many DNA copies - 4 categories of test: o Mutation scanning – is there any mutation present? o Mutation screening – is a known mutation present? o Genomic quantification – is there extra/missing DNA? o Sequencing – what is the exact DNA/RNA sequence? Hybridisation - Sense (coding) and antisense (template) strands are hybridised to each other in a dsDNA molecule - When heated to 95°C the strands denature (break apart) - Complimentary sequences can then hybridise to each strand Amplification (PCR) - Primer sequences can hybridise (anneal) to each strand at 5560°C - In the presence of dNTPs and DNA polymerase the complimentary DNA strand is extended in each direction (at 72°C) - The resulting 2 amplicons have the same sequence (copies) - Each cycle doubles the number of amplicons - amplification Exponential amplification of the target DNA sequence Mutation Scanning Methods - Is there any mutation present? - Examines a gene for all possible mutations (including unknown) - Based on analysis of physical or chemical characteristics of PCR product (hybridisation and amplification) - Abnormalities must be confirmed by sequencing - Methods include: o SSCP (single-strand conformation polymorphism) o DGGE (denaturing gradient gel electrophoresis) SSCP and DGGE Mutation Screening Methods - Is a known mutation present? - PCR based screening for known specific mutations - Indicated in diseases with few discrete, but frequent mutations - Need to know sequence you are looking for to design specific primers - Can check presence/absence and size of specific sequence of 50bp to 5kb (RFLP) - Methods include: o PCR o OLA o Southern Blotting/RFLP Polymerase Chain Reaction - Detects specific known mutations - Can check presence/absence and size of PCR product Allele-specific PCR and the oligonucleotide ligation assay Southern Blotting/RFLP Detects large scale changes in DNA fragments e.g. long repeat extensions in triplet repeat disorders Genomic quantification methods - Is there extra/missing DNA? - Can look at whole genome on one microarray - Detect duplications, heterozygous deletions, copy number variability, unbalanced structural abnormality at resolution of 30kb - Indicated in cases of multiple malformations or unknown dysmorphism - Limitations of arrays – can’t detect balanced rearrangements (inversions and translocations), small deletions or duplications, point mutations, expanded trinucleotide repeats. Not suitable for most monogenic disorders. - Methods: o DNA Microarrays o SNP arrays (SNP chips) DNA Microarray Analysis (CGH) Di-deoxy (Sanger) Sequencing Next Generation Sequencing (NGS) - Massively parallel sequencing technologies - Ultra-high throughput - High accuracy and flexibility - Dramatically cheaper than Sanger - Large range of “omics” applications o Metagenomics o Proteomics o Epigenomics Next Generation Sequencing A Word of Caution - Molecular genetic testing can provide a lot of information, both wanted and potentially unwanted - Need to consider how this could affect the patient, especially with regards to predictive testing - Sensitivity (true positive rate) and specificity (true negative rate) are important factors to consider when choosing a test. Things to Remember 1. Molecular Genetics refers to all diagnostic methods that examine changes of genetic information in extracted DNA or RNA 2. DNA array analysis relies on the principle of hybridisation and is useful for detecting any small unbalanced structural abnormality and numerical abnormalities 3. Next Generation Sequencing technologies hold promise for the diagnosis, prognosis and therapy of genetic diseases 4. Genetic testing can provide a lot of information – need to consider what is necessary information for the patient when choosing the test Question: What is the best type of molecular genetic test to do for a foetus with suspected Sickle Cell Disease? - A mutation screening test such as allele specific PCR or OLA - Sequencing of the β globin gene - A mutation scanning test such as SSCP - A genomic quantification test such as a DNA array