Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting...

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)