Nucleic Acid Methods PDF

Methoden der Biochemie 2 Nucleic acids Julian Stingele Fabio Spada Nucleic acid purification: extraction with organic solvents Aqueous Mix thoroughly with...

Methoden der Biochemie 2 Nucleic acids Julian Stingele Fabio Spada Nucleic acid purification: extraction with organic solvents Aqueous Mix thoroughly with an equal volume of organic solvent Centrifuge Collect aqueous phase e.g. phenol, chloroform, or phenol:chloroform Interphase Organic Perform additional extractions for increased purity Crude lysate containing The aqueous phase contains water-soluble molecules, Under acidic conditions RNA remains in the nucleic acids and other cell including nucleic acids. Proteins and lipids become trapped in aqueous phase, while DNA partitions into the constituents the organic phase, and are thus separated away. Insoluble interphase. This method was first established by debris becomes trapped in the interphase between the two Chomczynski and Sacchi ,1987, and underlies layers many current commercial products for RNA isolation (RNAzol, TRIzol, etc.). Nucleic acid purification: spin column chromatography with silica matrix Lysis with chaotropic salt (guanidinium) Removing unwanted nucleic acids Add DNase + DNase (protein) Add RNase + RNase (protein) Depending on when nuclease treatment is performed and/or on downstream applications, it may be necessary to repeat purification steps for protein removal (e.g. phenol/chloroform extraction). Nucleic acid quantitation: Spectrophotometry - NanoDrop By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak at ~260nm. [dsDNA] ≈ A260 x (50 µg/mL) Oligonucleotides - if extinction coefficient at λ = 260 nm (ε260) and light path length (L) are given: [ssDNA] ≈ A260 x (33 µg/mL) Molar concentration [M] can be calculated through the Lambert-Beer Law: A260 = [M] x ε260 x L [ssRNA] ≈ A260 x (40 µg/mL) Nucleic acid quantitation using fluorescent dyes: Fluorometry (e.g., Qubit) DNA/RNA-selective fluorescent dye Nucleic acid quality control: RNA Number of genes for each RNA Transcriptional activity RNA abundance polymerase Pol II 13% Pol II Ribo 13% Pol III Pol I 14% 60% Warner TIBS 1999 - Li…Willis MCB 2000 Nucleic acid quality control: RNA Ribosomal RNA (rRNA) makes up more than 80% of total RNA samples. Total RNA preps should display two prominent bands after gel electrophoresis. Nucleic acid quality control: chip-electrophoresis (e.g., BioAnalyzer, TapeStation) Nucleic acid visualization/detection: Fluorescent dyes: EtBr, SYBR family, … Nucleic acid visualization/detection: northern/Southern blot or Nylon filter northern blot: RNA detection Expression profiles in various tissues Southern blot: DNA detection Genotyping to verify successful gene targeting Fluorescent in situ hybridization: FISH Nucleic acid visualization/detection: Fluorescent in situ hybridization Chromosome painting can be applied as diagnostic method for the detection of chromosome translocations Detection of a chromosome 10:17 translocation (white arrows point at the products of a full arm reciprocal translocation) t10:17 Chr. 17 t10:17 Chr. 10 Modern molecular cytogenetic techniques in genetic diagnostics: https://doi.org/10.1016/S1471-4914(02)02335-3 PCR 1st PCR cycle 2nd PCR cycle D Template DNA Denaturation Primer annealing Primer extension e Primer annealing Primer extension n a t u r a t i o n The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—denaturation, annealing, and elongation—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules The key to PCR are heat-stable DNA polymerases such as Taq polymerase (from Thermus aquaticus) and others from different thermophilic bacteria. Generating cDNA DNA in nucleus mRNAs in cytoplasm Reverse mRNA transcriptase Poly(A) tail A A A A A A 3’ 3 T T T T T 5’ DNA Primer strand A A A A A A 3’ T T T T T 5’ 3  5’ DNA polymerase 3’ 5’ cDNA Quantitative / real-time PCR Quantitative / real-time PCR: SyBr Green Product can be further tested in a post-amplification Binds minor groove of double-stranded DNA. melting curve in which sequences have characteristic melting temperatures. Sanger sequencing https://en.wikipedia.org/wiki/Sanger_sequencing Next (second) generation sequencing: Solexa/Illumina sequencing https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced/ https://www.youtube.com/watch?v=0vqajoP08Jg https://www.youtube.com/watch?v=CZeN-IgjYCo Next (second) generation sequencing: Solexa/Illumina sequencing https://www.youtube.com/watch?v=0vqajoP08Jg https://www.youtube.com/watch?v=CZeN-IgjYCo Second generation sequencing: Bridge amplification Second generation sequencing: Cluster generation Second generation sequencing: clonal single molecule array ~ 100 µm2 cluster 100 µm Second generation sequencing: sequencing by synthesis 2nd gen. sequencing is short read sequencing: The efficiency of these chemical reactions is limited: The maximal size of fragments that can be fully sequenced is ~150 bp for highly complex libraries and ~300 for less complex ones. The first step in library preparation is shearing of DNA/RNA fragments down to an optimal size. Second generation sequencing Second generation sequencing Fragment shearing + Third generation sequencing technologies: Nanopore sequencing Instruments: PromethION GridION MinION Helicase (motor protein) Advantages of Nanopore sequencing: Very long reads: high processivity of unwinding enzyme / no limiting reagent. Much improved haplotype assembly (long stretches of chromosomal DNA). Characterisation of alternative mRNA splicing events. If DNA/RNA sample relatively abundant, no need of amplification (no amplification artefacts). (synthetic copolymer) Direct identification of modified bases 5mC & 6mA without chemical treatments (epigenomics & epitranscriptomics). Disadvantages of Nanopore sequencing: Quality and integrity of NA library are limiting factors (pore clogging). Relatively high error rates in calling of modified bases. (new algorithms and AI are improving modified base identification). DNA - protein interaction: Chromatin immuno-precipitation - ChIP Cell are crosslinked with formaldehyde Immunoprecipitation with antibody Purification of copurifying DNA DNA detection by PCR / sequencing DNA - protein interaction: Chromatin immunoprecipitation-seq (ChIP-seq)