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Louise Stanley Newcastle

Describe at least 5 methods of scanning for unknown point mutations. From a diagnostic laboratories perspective, highlight the underlying principles and relative advantages and disadvantages of each method. Louise Stanley Newcastle. Keywords. Mutation Detection Sequencing

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Louise Stanley Newcastle

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  1. Describe at least 5 methods of scanning for unknown point mutations. From a diagnostic laboratories perspective, highlight the underlying principles and relative advantages and disadvantages of each method. Louise Stanley Newcastle

  2. Keywords • Mutation Detection • Sequencing • Heteroduplex analysis – HRM, dHPLC, CSCE • PTT • Microarrays

  3. Definition • Mutation scanning techniques are used to detect sequence variants without the need for prior knowledge of the identity or precise location of the variant, in contrast with genotyping techniques, which determine the status of a specific variant.

  4. Direct Analysis • No prior pre-screen - provides direct information on the sequence variant identified with no need for further investigation • Gold Standard – fluorescent Sanger Sequencing • Number of different genetic analysers available with different numbers of capillaries – increased number of capillaries = increased throughput (1, 4, 8, 16, 24, 48 and 96)

  5. Sanger Sequencing • Principles: • Target specific primers designed to amplify coding exons and flanking intronic regions - usually designed with universal sequencing tag (e.g. M13) to enable streamlined downstream processing post PCR. • Sequencing primers bound to ss template DNA and dNTPs are added by polymerase (in 5’-3’ direction). (Separate F and R reactions) • Fluorescently labelled chain terminating ddNTPs (lack 3’ hydroxyl group preventing chain elongation), present in limited quantities so on average each position of the target template is probed once. • Products analysed and separated according to size using fluorescent capillary electrophoresis (thin capillary contain polyacrylamide gel which maintains DNA in single strand state – denaturing conditions). • Four differently labelled ddNTPS enables reaction to take place in one tube (different emission wavelengths).

  6. Sanger Sequencing • Difference between dATP and ddATP CHAIN TERMINATING

  7. Sanger Sequencing • Advantages: • Direct analysis – information provided directly on sequence change • High sensitivity • Easily automated – both PCR and sequence set up • Can easily analyse genes with low sample numbers, also suited for high throughput analysis • Disadvantages • Expensive – although costs coming down • Identify variants with uncertain clinical significance making interpretation of results difficult • Data analysis time consuming although becoming more automated • Starting DNA quality has to be good – difficult when using tumour or archived material • Sequence output limited to ~30-60kb of sequence per 3-4 hour run

  8. Next-Generation Sequencing • Principles – discussed this morning – different platforms available: e.g. Roche 454, Illumina, ABI-Solid but all have very large sequence capacity outputs compared to traditional ABIs • Advantages: • Ability to examine multiple targets • Increased throughput – potential to examine increased regions of the genome – i.e. promoter and deep intronic regions. Increased capacity may allow more patients to be examined – e.g. low and medium risk BRCA patients who would otherwise not get screening • Target entire disease pathways apposed to individual genes • Reduced reagent/running costs providing capacity optimised • Disadvantages: • Very Expensive equipment • Most diagnostic laboratories limited access to machines • Technically demanding process • Huge amount of data produced • Data storage • Specialist bioinformatics • Expensive commercialised software • Exome Sequencing – ability to identify pathogenic mutations without any prior knowledge of candidate gene(s) – currently beyond the capabilities/capacity of diagnostic laboratories

  9. Indirect/Pre-screening Methods • Used in general to reduce pressure on equipment used for sequencing and cheaper/quicker than direct sequence analysis • Many examples exploit the formation of heteroduplexes: • Most mutations occur in heterozygous form (exceptions – X-linked disorders in males, offspring of consanguineous marriages). • Heteroduplexes can be formed by simply heating the PCR-amplified DNA to 94oC and slowly cooling it. • For homozygous mutations or for X-linked mutations in males it is necessary to add some reference wild-type DNA - additional step in process.

  10. Heteroduplex Analysis • General Advantages: • Cheaper than sequencing • Quicker • Data analysis usually easier and less time consuming • General Disadvantages: • Doesn’t provide direct information on sequence change – those showing differences from WT require sequencing • Will identify polymorphisms which adds to subsequent sequence load, therefore not very suitable for highly polymorphic genes • Can require additional expensive equipment – e.g. WAVE machine, LightScanner

  11. HRM - LightScanner • HRM – High Resolution Melt Curve Analysis • Principle: PCR carried out in presence of saturating dsDNA binding dye such as LCGreen – Form heteroduplexes by denaturing and slowly cooling products. Carry out melt curve analysis - dye dissociates as product is denatured resulting in a change in fluorescence – those with mismatches are more unstable and dye dissociates at a lower temperature therefore get differences in melt curve to WT samples – software can present data as easily interpretable difference curves An example showing the difference between the melt curves from a WT and a fragment containing a nucleotide change (www.idahotech.com).

  12. HRM - LightScanner • Advantages: • Entire process very quick – 1.5 hour PCR program followed by 5 min data collection on machine and analysis of results very quick • High sensitivity (providing optimal optimisation) • Reagents cheap • Closed tube system – post PCR doesn’t require any additional steps therefore risk of contamination reduced • Analysis is non-destructive and therefore following analysis products can be directly sequenced • Disadvantages: • Not suitable for highly polymorphic genes – although specific probes can be designed to reduce this work load • Fragment size is small (~150-200bp optimum) • Small indels, particularly in low complexity regions, are the most difficult to detect by HRM – may miss these mutations. • HRM has more stringent requirements in terms of the location of primers to limit melting domains and may require more fragment specific optimisation with positive controls to ensure suitable melting and to set the analysis parameters. Too many melt domains and will loose sensitivity • Best suited for analysing large number of samples to enable accurate data analysis. Fewer sample numbers lead to poorer results and greater number of samples requiring sequencing. • Analysis best suited to DNAs extracted by same method – problematic if offering service to other diagnostic laboratories as it makes batching difficult • Analysis not so easily automated compared with sequence analysis – some changes can be very subtle therefore need expertise in interpretation

  13. CSCE • Conformation sensitive capillary electrophoresis • Fluorescent based heteroduplex analysis • Changes in mobility • Run under partial denaturating conditions Complete resolution Incomplete resolution

  14. CSCE • Advantages: • Sensitive – able to detect very subtle changes in profiles • Less complex analysis than sequencing (short analysis time) • Different fluorescent labels enables multiplexing (although fairly limited) • Disadvantages: • Most likely to miss single base substitutions • Requires modified conformational analysis polymer • Machine used for CSCE cannot be used for sequencing – need to change array • May need post PCR processing to dilute samples

  15. dHPLC – WAVE Machine • Denaturing high-performance liquid chromatography • Principles: • Wild-type reference fragment and patient samples are amplified separately. • Heteroduplex formation. • Sample is injected into a flow path of TCA (Acetonitrile) and TEAA (Triethylammonium acetate) • The temperature within the chamber is set to enable partial denaturation • The hydrophobic portion of TEAA interacts with the hydrophobic beads in the cartridge • The samples which are injected into the flow path then bind to the beads through the interaction of the negatively charged phosphate backbone of the partially denatured fragments to the positively charged ammonium groups of TEAA • At increasing concentrations of acetonitrile the TEAA/DNA attraction is reduced and the DNA fragments begin to elute off the cartridge. • The heteroduplexes with the mismatched base pairs at a site of a mutation elute first, followed by the homoduplexes • A UV detector measures absorbance as the fragments pass through.

  16. dHPLC – WAVE Machine Absorbance Time

  17. dHPLC – WAVE Machine • Advantages: • Doesn’t require modified primers or detection labels • Cheaper than sequencing • Sensitive • Disadvantages: • Requires expensive equipment • Toxic waste chemicals • Run time of machine slow – amplicons often need analysing at two different temperatures • Machine requires regular maintenance

  18. Microarrays • Principle: • Custom microarrays can interrogate every position in a gene in one assay. • Use either amplified cDNA or exons from amplified genomic DNA hybridised to microarray containing overlapping oligonucleotides corresponding to every part of the sequence. • Short oligonucleotides require exact matching sequence to hybridise efficiently. • Probes are organised into sets that query both the forward and reverse stands. • Affymetrix system uses 40 probes (each 24 nucleotides in length) per nucleotide. • Base calling based on algorithms that compare hybridisation intensities of all 40 probes.

  19. Microarrays • Advantages: • Good for initial scanning of samples to pick up common mutations – direct information provided on mutation • Disadvantages: • Not suitable for large deletions/insertions • Range of mutations has to be defined in advance and designed onto chip • Reagents costs high • Expensive specialised equipment required • Technical expertise

  20. PTT – Protein Truncation Test • Largely outdated method • Principle: Based upon an in vitro-coupled transcription and translation of PCR amplified coding sequences also referred to as in vitro synthesised protein (IVSP) • Isolation of genomic DNA or RNA from blood or tissue samples. • Amplification of target sequence using PCR if the sample being used is genomic DNA or by reverse transcription PCR (RT-PCR) if RNA is being used. • Products used for the in vitro transcription and translation which can be carried out quickly by the use of the TNT kit from Promega. • SDS-PAGE for protein analysis. The shorter protein products of the mutated alleles can be easily distinguished from the full length protein products of the normal alleles. Most commonly achieved by incorporation of radioactive amino acids during protein synthesis such as 35S methionine, 35S cysteine or 3H leucine

  21. PTT – Protein Truncation Test • Advantages: • Only identifies truncating (premature termination codons and frameshifts) - mutations – doesn’t detect missense UCVs that can be difficult to interpret. Suited to genes where majority are truncating eg. BRCA1 exon 10 (3.4kb), APC exon 15 (6.5kb) [genomic DNA rather than RNA can be analysed in these cases] • Large coding regions can be covered in one fragment which would otherwise require breaking down into much smaller sections by sequence analysis • Gives approximate location of mutation • Equipment costs fairly minimal but reagents expensive? • Disadvantages: • Technical process • Requires radioactivity • Will not detect pathogenic missense mutations • Time consuming – not very robust method, largely outdated • Small number of samples analysed at once – not appropriate for high throughout analysis

  22. Other methods • Chemical/Enzymatic mismatch cleavage of heteroduplexes • SSCP • MALDI-TOF

  23. References: • Strachan and Read, Molecular Genetics Edition 4 • http://www.ngrl.org.uk/wessex/ • www.idahotech.com/LightScanner/ • http://www.transgenomic.com/ap/VariationApp1.asp

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