techniques of molecular biology. basic molecular biology techniques isolating nucleic acids cutting...
TRANSCRIPT
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 agaroseor 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
The polymerase chain reaction (PCR)
DNA polymerasesdATP dTTP dGTP dCTP
DNA polymerases
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
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 ofthe strands
Sequencing techniques
• dideoxysequencing• pyrosequencing
Sequencing techniques
• dideoxysequencing
Sequencing techniques
• dideoxysequencing
≈ 800 nucleotidescan be sequencedin one run
polyacrylamide gelelectrophoresis
Sequencing techniques
• dideoxysequencing• pyrosequencing
≈ 200 nucleotidescan be sequencedin 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 genomesSplit 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
gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgttttaagattaataaaattatgtctaaccaagtatttactactttacgcgcagcaacattagctgttattttaggtatggctggtggcttagcagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtgaggctaatggtcgtattgtatgtgcaaactgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttccatacgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttgaattagcaccaccagatcgcgttccggcagaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaatattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacctattttatctccagatcctgctaaaaataaaaacgtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaacactatttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagcaaacggtgaagttgttgtagacaaaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccattaacaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaattgtattacaaaaccctgctcgtattcaaggtttattagtattcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatgaacttctaatatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttctacttgaacgatatttagcttttaatgcTATAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatgtaaacctgctaacgatatttatatataagcattttaatactccgagggaggcctctaacctttagcaagtaagtaaacttccccttcggggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatgcaaagcatggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacatactccgaaggaggacgtcagtggcagtggtaccgccactgctattttaatactccgaaggagcagtggtggtcccactgccactaaaatttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtcagtggcagttgcctgccaactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctccttccccttcgggcaagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaatttgtccccttcgggcaaataaatcttagtggcagttgcaaaatattaatatcgtatataaatttggagtatataaataaatttggagtatataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcccgaaggggacgtcctgccaactgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgtccccttcgggt
Expasy server
Finding open reading frames
Sequence from the E. coli genome
The E. coli genome
5’ UTR 3’ UTRcoding region = open reading frames
Translation start
Translation stop
5’ - - 3’
protein-coding gene = DNA transcribed into mRNA
Protein-coding genes
Genes = all DNA sequences that are transcribed into RNA
UTR = untranslated region
Figure 5.4 Genomes 3 (© Garland Science 2007)
Exons and introns in eukaryotic genes
5’ UTR 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)
Figure 5.10 Genomes 3 (© Garland Science 2007)
A typical sequence annotation result
Verifying the identity of a gene
• Homology search
• Experimental techniques
Northern hybridizationZoo-blotting
Verifying the identity of a gene
• Homology search
MSNQVFTTLR AATLAVILGM AGGLAVSPAQ AYPVFAQQNY ANPREANGRI VCANCHLAQK AVEIEVPQAV LPDTVFEAVI ELPYDKQVKQ VLANGKKGDL NVGMVLILPE GFELAPPDRV PAEIKEKVGN LYYQPYSPEQ KNILVVGPVP GKKYSEMVVP ILSPDPAKNK NVSYLKYPIY FGGNRGRGQV YPDGKKSNNT IYNASAAGKI VAITALSEKK GGFEVSIEKA NGEVVVDKIP AGPDLIVKEG QTVQADQPLT NNPNVGGFGQ AETEIVLQNP ARIQGLLVFF SFVLLTQVLLVLKKKQFEKV QLAEMNF
BLAST
BLAST = Basic Local Alignment Search Tool
Figure 5.28 Genomes 3 (© Garland Science 2007)
6274 ORFsCase study, yeast genome
Finding the function of a gene (product)
Computer based analysis
Homology search
Experimental analysis
Gene inactivationOverexpression
Whole genome studies
Tiling assays
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
Liquid chromatography is used to separatepeptides before mass spectrometry
Mass spectrum
Mass spectra are compared to theoretical values
Figure 6.11 Genomes 3 (© Garland Science 2007)
Mouse liver proteins
Figure 6.20a Genomes 3 (© Garland Science 2007)
Protein interaction map of yeast
Nucleic acid protein interactions
Electrophoretic mobilityshift assay (EMSA)
Nuclease protection footprinting
In vitro selectionassay
Chromatin immuno-precipitation (ChIP)
Chromosome conformation capture (3C assay)