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 vectorGenomic librarycDNA 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 polymerases dATP 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 of the strands

Sequencing techniques dideoxysequencing pyrosequencing

Sequencing techniques dideoxysequencing

Sequencing techniques dideoxysequencing ≈ 800 nucleotides can be sequenced in one run polyacrylamide gel electrophoresis

Sequencing techniques dideoxysequencing pyrosequencing ≈ 200 nucleotides can be sequenced in one run

Next generation sequencing methods

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

gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgttttaagattaataaaattatgtctaaccaagtattta ctactttacgcgcagcaacattagctgttattttaggtatggctggtggcttagcagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtga ggctaatggtcgtattgtatgtgcaaactgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttccata cgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttgaattagcaccaccagatcgcgttccggca gaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaatattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacc tattttatctccagatcctgctaaaaataaaaacgtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaaca ctatttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagcaaacggtgaagttgttgtagaca aaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccattaacaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaatt gtattacaaaaccctgctcgtattcaaggtttattagtattcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatga acttctaatatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttctacttgaacgatatttagcttttaatgcTA TAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatgtaaacctgctaacgatatttatatataagcattttaatactccgagggaggcctct aacctttagcaagtaagtaaacttccccttcggggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatg caaagcatggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgt ccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacatactccgaaggaggacgtcagtggcagtggtaccgccactgctatttta atactccgaaggagcagtggtggtcccactgccactaaaatttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtc agtggcagttgcctgccaactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctccttccccttcgggc aagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaatttgtccccttcgggcaaataaatcttagtggcagttgcaa aatattaatatcgtatataaatttggagtatataaataaatttggagtatataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcc cgaaggggacgtcctgccaactgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgtccccttcgggt Expasy server Finding open reading frames

Sequence from the E. coli genome

The E. coli genome

5’ UTR3’ UTR coding 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’ UTR3’ 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 hybridization Zoo-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 SFVLLTQVLL VLKKKQFEKV 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 inactivation Overexpression

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 separate peptides 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 mobility shift assay (EMSA)

Nuclease protection footprinting

In vitro selection assay

Chromatin immuno- precipitation (ChIP)

Chromosome conformation capture (3C assay)