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Techniques of Molecular Biology Basic molecular biology techniques • • • • • Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques Genomics • Sequencing genomes • Analyzing genome sequences Proteomics • Separating proteins • Analyzing proteins Basic molecular biology techniques • Isolating nucleic acids Basic molecular biology techniques • Isolating nucleic acids • Cutting DNA into fragments DNA can be reproducibly split into fragments by restriction endonucleases DNA fragments can be separated by size in agarose or polyacrylamide gels Because of the phosphates in the sugar phosphate backbone, nucleic acids are negatively charged. In an electric field nucleic acids will move towards the positive pole. Smaller fragments move faster than larger fragments through the pores of a gel. Basic molecular biology techniques • Isolating nucleic acids • Cutting DNA into fragments • Ligating DNA fragments Basic molecular biology techniques • • • • Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments DNA can be amplified by • Cloning • PCR DNA cloning and construction of DNA libraries Cloning in a plasmid vector Genomic library cDNA library Vectors for DNA cloning Basic molecular biology techniques • • • • Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments DNA can be amplified by • Cloning • PCR DNA polymerases dATP dTTP dGTP dCTP DNA polymerases The polymerase chain reaction (PCR) Basic molecular biology techniques • • • • • Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques Single-stranded nucleic acids can bind to each other by base pairing if they contain complementary sequences Using a single-stranded labeled probe complementary base pairing is able to detect specific nucleic acids among many different nucleic acids. If the probe is used to detect DNA, the analysis is called DNA blot (Southern) analysis. If an RNA fragment is detected, the analysis is called RNA blot (northern) analysis. Transcriptome analysis using microarrays 26x24 = 624 spots Basic molecular biology techniques • • • • • Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques Genomics • Sequencing genomes Sequencing techniques • • dideoxysequencing pyrosequencing dATP dTTP dGTP dCTP Genomic library • denature (make single-stranded) • anneal primer extend primer to copy one of the strands Sequencing techniques 2’-3’-dideoxynucleotide 2’ deoxynucleotide • • dideoxysequencing pyrosequencing Base Base Sequencing techniques 2’-3’-dideoxynucleotide 2’ deoxynucleotide • dideoxysequencing Base Base ddCTP Sequencing techniques • ddTTP ddGTP dideoxysequencing polyacrylamide gel electrophoresis ≈ 800 nucleotides can be sequenced in one run Sequencing techniques • • dideoxysequencing pyrosequencing ≈ 200 nucleotides can be sequenced in one run Next generation sequencing methods https://en.wikipedia.org/wiki/DNA_sequencing Genomics • Sequencing genomes (assembling the sequence) Genomics • Sequencing genomes (assembling the sequence) Genomics • Sequencing genomes • (assembling the sequence) Genomics • Sequencing genomes • Analyzing genome sequences Genomics • Sequencing of genomes Split genome into pieces and sequence all pieces. Assembling the sequence (computer). • Sequence analysis (annotation 1) Identify genes and other elements in sequence. • Functional analysis (annotation 2) Determine function of identified elements. How to find genes in a genome sequence Protein-coding genes • • • • Find open reading frames (protein-coding sequences) Find sequence with a codon bias Find upstream regulatory sequences (e.g. CpG islands) Find exon-intron boundaries Genes coding for functional RNAs • Find consensus sequences for tRNAs and ribosomal RNAs • Find specific RNA secondary structures (e.g. stem loops) • Find upstream regulatory sequences Genomic sequence Finding open reading frames Finding open reading frames gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgtttt aagattaataaaattatgtctaaccaagtatttactactttacgcgcagcaacattagctgttattttaggtatggctggtggcttag cagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtgaggctaatggtcgtattgtatgtgcaaa ctgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttcca tacgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttg aattagcaccaccagatcgcgttccggcagaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaa tattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacctattttatctccagatcctgctaaaaataaaaac gtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaacacta tttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagc aaacggtgaagttgttgtagacaaaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccatta acaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaattgtattacaaaaccctgctcgtattcaaggtttattagtat tcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatgaacttcta atatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttct acttgaacgatatttagcttttaatgcTATAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatg taaacctgctaacgatatttatatataagcattttaatactccgagggaggcctctaacctttagcaagtaagtaaacttccccttcg gggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatgcaaagca tggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattt taatgcaataaataaatttgtccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacat actccgaaggaggacgtcagtggcagtggtaccgccactgctattttaatactccgaaggagcagtggtggtcccactgccactaaaa tttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtcagtggcagttgcctgc caactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctcctt ccccttcgggcaagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaat ttgtccccttcgggcaaataaatcttagtggcagttgcaaaatattaatatcgtatataaatttggagtatataaataaatttggagt atataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcccgaaggggacgtcctgcca actgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgt ccccttcgggt Expasy server Sequence from the E. coli genome The E. coli genome Genes = all DNA sequences that are transcribed into RNA Protein-coding genes 5’ UTR coding region = open reading frames 3’ UTR 5’ - - 3’ Translation start Translation stop protein-coding gene = DNA transcribed into mRNA UTR = untranslated region Exons and introns in eukaryotic genes 5’ UTR Figure 5.4 Genomes 3 (© Garland Science 2007) 3’ UTR How to find genes in a genome sequence Protein-coding genes • • • • Find open reading frames (protein-coding sequences) Find sequence with a codon bias Find upstream regulatory sequences (e.g. CpG islands) Find exon-intron boundaries Genes coding for functional RNAs • Find consensus sequences for tRNAs and ribosomal RNAs • Find specific RNA secondary structures (e.g. stem loops) • Find upstream regulatory sequences Figure 5.6b Genomes 3 (© Garland Science 2007) A typical sequence annotation result Figure 5.10 Genomes 3 (© Garland Science 2007) Verifying the identity of a gene • Homology search • Experimental techniques Northern hybridization Zoo-blotting Verifying the identity of a gene • Homology search MSNQVFTTLR VCANCHLAQK NVGMVLILPE GKKYSEMVVP IYNASAAGKI QTVQADQPLT VLKKKQFEKV AATLAVILGM AVEIEVPQAV GFELAPPDRV ILSPDPAKNK VAITALSEKK NNPNVGGFGQ QLAEMNF BLAST AGGLAVSPAQ LPDTVFEAVI PAEIKEKVGN NVSYLKYPIY GGFEVSIEKA AETEIVLQNP BLAST = Basic Local Alignment Search Tool AYPVFAQQNY ELPYDKQVKQ LYYQPYSPEQ FGGNRGRGQV NGEVVVDKIP ARIQGLLVFF ANPREANGRI VLANGKKGDL KNILVVGPVP YPDGKKSNNT AGPDLIVKEG SFVLLTQVLL Case study, yeast genome Figure 5.28 Genomes 3 (© Garland Science 2007) 6274 ORFs Finding the function of a gene (product) Computer based analysis Homology search Experimental analysis Gene inactivation Overexpression Whole genome studies Tiling assays Proteomics • Isolating and separating proteins • Identifying and analyzing proteins Working with proteins • Separating proteins • Analyzing proteins and their interactions Separating proteins on polyacrylamide gels Immunoblot (Western blot) Proteins can be sequenced Complex mixtures of proteins can be analyzed by mass spectrometry Typical workflow in analysis of proteins by mass spectometry Liquid chromatography is used to separate peptides before mass spectrometry Mass spectrum Mass spectra are compared to theoretical values Mouse liver proteins Figure 6.11 Genomes 3 (© Garland Science 2007) Protein interaction map of yeast Figure 6.20a Genomes 3 (© Garland Science 2007) Nucleic acid protein interactions Electrophoretic mobility shift assay (EMSA) Nuclease protection footprinting is used to identify the DNA sequence to which a protein binds In vitro selection assay uses a combinatorial DNA sequence library to identify DNA sequences to which a protein binds. Chromatin immunoprecipitation (ChIP) Identifies protein-binding sites in vivo Chromosome conformation capture (3C assay) Identifies DNA sequences that are conformationally linked