Chapter 2 The Extraction of DNA and RNA and Basic knowledge.ppt

Chapter 2The Extraction of DNA and RNAThe extraction of DNA1. 1.Ensure the integrity of the DNA Ensure the integrity of the DNA structurestructure2.2.Try to eliminate the pollution of other Try to eliminate the pollution of other macromolecular components (such as macromolecular components (such as protein, polysaccharide, RNA, etc.)protein, polysaccharide, RNA, etc.)3.3.Ensure that the extracted sample does Ensure that the extracted sample does not contain any organic solvent or high not contain any organic solvent or high concentration of metal ions that can concentration of metal ions that can inhibit the enzymesinhibit the enzymesAttentions Basic stepsvSampling (as fresh as possible)vGrinded with liquid nitrogen vLysisvCentrifugation to remove protein and cell debrisvThe resulting DNA solution is concentratedvWashed with 70% alcohol (23 times)vDissolved with TE Buffer of ddH2OThe extraction of RNAThe extraction of RNA-Trizol method Application:Human, Animals, Plants, The tissue or Application:Human, Animals, Plants, The tissue or culture of microorganismsculture of microorganisms (mg-g)(mg-g)vvAdvantageAdvantage：No protein and DNA pollution.No protein and DNA pollution.vvUse Use ：1. 1.NorthernNorthern blotblot，Dot-blot hybridizationDot-blot hybridization，Poly(A)+separation.Poly(A)+separation.2.2.RNase blocking analysis of translation in vitro.RNase blocking analysis of translation in vitro.3.3.Molecular cloning.Molecular cloning.Determination of DNA and RNA concentration and purity judgmentOD260/OD280 1.8 May be polluted by RNAOD260/OD280 1.8 May be polluted by proteinsPure DNA：OD260/OD280=1.8Pure RNA：OD260/OD280=2.0vTransitionvTransversionvSynonymous substitutionvNonsynonymous substitutionvCodon usage bias vNonsense mutationSeveral easily confused conceptions Transition/transversionTransition, in genetics and molecular biology, refers to a point mutation that changes a purine nucleotide to another purine (A G), or a pyrimidine nucleotide to another pyrimidine (C T). Approximately two out of three single nucleotide polymorphisms (SNPs) are transitions.Transversion, in molecular biology, refers to a point mutation in deoxyribonucleic acid (DNA), where a single (two ring) purine is changed for a (one ring) pyrimidine, or vice versa.Degeneracy within the Universal Genetic code. Silent and Replacement SubstitutionsvSilent substitution:Sequence 1: UUU CAU CGUSequence 2: UUU CAC CGU Coded Amino Acids: Phe His Arg lReplacement substitution:Sequence 1: UUU CAU CGUSequence 2: UUU CAG CGU Coded Amino Acids: Phe His Arg GlnSilent and Replacement SubstitutionsvSilent substitution:Sequence 1: UUU CAU CGUSequence 2: UUU CAC CGU Coded Amino Acids: Phe His Arg lReplacement substitution:Sequence 1: UUU CAU CGUSequence 2: UUU CAG CGU Coded Amino Acids: Phe His Arg GlnNegative versus Positive selectionvNegative selectionVery few Replacement substitutionsNo difference expected whether fixed or not. Ratios the same.vPositive selectionReplacement substitutions that are beneficial are kept within the populationMore Fixed than Polymorphic replacement substitutions Ratios differ.How do you detect positive selection?vDnNumber of replacement substitutions Number of replacement sitesvDsNumber of silent substitutions Number of silent sitesvDn/Ds 1 Positive SelectionCodon usage bias refers to differences in the frequency of occurrence of synonymous codons in coding DNA. A codon is a series of three nucleotides (a triplet) that encodes a specific amino acid residue in a polypeptide chain or for the termination of translation (stop codons). There are 64 different codons (61 codons encoding for amino acids plus 3 stop codons) but only 20 different translated amino acids. The overabundance in the number of codons allows many amino acids to be encoded by more than one codon. The genetic codes of different organisms are often biased towards using one of the several codons that encode the same amino acid over the othersthat is, a greater frequency of one will be found than expected by chance.Codon usage bias It is generally acknowledged that codon biases reflect a balance between mutational biases and natural selection for translational optimization. Optimal codons in fast-growing microorganisms, like Escherichia coli or Saccharomyces cerevisiae (bakers yeast), reflect the composition of their respective genomic tRNA pool. It is thought that optimal codons help to achieve faster translation rates and high accuracy. As a result of these factors, translational selection is expected to be stronger in highly expressed genes, as is indeed the case for the above-mentioned organisms. In other organisms that do not show high growing rates or that present small genomes, codon usage optimization is normally absent, and codon preferences are determined by the characteristic mutational biases seen in that particular genome.In genetics, a point-nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a point。