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Page 1: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Techniques of Molecular Biology

Page 2: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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

Page 3: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids

Page 4: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids• Cutting DNA into fragments

Page 5: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

DNA can be reproducibly split into fragments by restriction endonucleases

Page 6: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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.

Page 7: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids• Cutting DNA into fragments• Ligating DNA fragments

Page 8: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids• Cutting DNA into fragments• Ligating DNA fragments• Amplifying DNA fragments

DNA can be amplified by

• Cloning

• PCR

Page 9: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

DNA cloning and construction of DNA libraries

Cloning in a plasmid vector Genomic library cDNA library

Page 10: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Vectors for DNA cloning

Page 11: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids• Cutting DNA into fragments• Ligating DNA fragments• Amplifying DNA fragments

DNA can be amplified by

• Cloning

• PCR

Page 12: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

The polymerase chain reaction (PCR)

Page 13: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

DNA polymerasesdATP dTTP dGTP dCTP

Page 14: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

DNA polymerases

Page 15: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids• Cutting DNA into fragments• Ligating DNA fragments• Amplifying DNA fragments• Hybridization techniques

Page 16: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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.

Page 17: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Transcriptome analysis using microarrays

Page 18: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Basic molecular biology techniques• Isolating nucleic acids• Cutting DNA into fragments• Ligating DNA fragments• Amplifying DNA fragments• Hybridization techniques

Genomics• Sequencing genomes

Page 19: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Sequencing techniques

• dideoxysequencing• pyrosequencing

dATP dTTP dGTP dCTP

Genomic library

• denature (make single-stranded)• anneal primer

extend primer to copy one ofthe strands

Page 20: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Sequencing techniques

• dideoxysequencing• pyrosequencing

Page 21: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Sequencing techniques

• dideoxysequencing

Page 22: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Sequencing techniques

• dideoxysequencing

≈ 800 nucleotidescan be sequencedin one run

polyacrylamide gelelectrophoresis

Page 23: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Sequencing techniques

• dideoxysequencing• pyrosequencing

≈ 200 nucleotidescan be sequencedin one run

Page 24: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Next generation sequencing methods

https://en.wikipedia.org/wiki/DNA_sequencing

Page 25: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Genomics• Sequencing genomes (assembling the sequence)

Page 26: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Genomics• Sequencing genomes (assembling the sequence)

Page 27: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Genomics• Sequencing genomes • (assembling the sequence)

Page 28: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Genomics• Sequencing genomes• Analyzing genome sequences

Page 29: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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.

Page 30: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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

Page 31: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Genomic sequence

Page 32: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Finding open reading frames

Page 33: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgttttaagattaataaaattatgtctaaccaagtatttactactttacgcgcagcaacattagctgttattttaggtatggctggtggcttagcagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtgaggctaatggtcgtattgtatgtgcaaactgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttccatacgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttgaattagcaccaccagatcgcgttccggcagaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaatattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacctattttatctccagatcctgctaaaaataaaaacgtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaacactatttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagcaaacggtgaagttgttgtagacaaaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccattaacaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaattgtattacaaaaccctgctcgtattcaaggtttattagtattcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatgaacttctaatatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttctacttgaacgatatttagcttttaatgcTATAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatgtaaacctgctaacgatatttatatataagcattttaatactccgagggaggcctctaacctttagcaagtaagtaaacttccccttcggggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatgcaaagcatggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacatactccgaaggaggacgtcagtggcagtggtaccgccactgctattttaatactccgaaggagcagtggtggtcccactgccactaaaatttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtcagtggcagttgcctgccaactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctccttccccttcgggcaagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaatttgtccccttcgggcaaataaatcttagtggcagttgcaaaatattaatatcgtatataaatttggagtatataaataaatttggagtatataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcccgaaggggacgtcctgccaactgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgtccccttcgggt

Expasy server

Finding open reading frames

Page 34: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Sequence from the E. coli genome

Page 35: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

The E. coli genome

Page 36: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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

Page 37: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Figure 5.4 Genomes 3 (© Garland Science 2007)

Exons and introns in eukaryotic genes

5’ UTR 3’ UTR

Page 38: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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

Page 39: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Figure 5.6b Genomes 3 (© Garland Science 2007)

Page 40: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Figure 5.10 Genomes 3 (© Garland Science 2007)

A typical sequence annotation result

Page 41: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Verifying the identity of a gene

• Homology search

• Experimental techniques

Northern hybridizationZoo-blotting

Page 42: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

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

Page 43: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Figure 5.28 Genomes 3 (© Garland Science 2007)

6274 ORFsCase study, yeast genome

Page 44: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Finding the function of a gene (product)

Computer based analysis

Homology search

Experimental analysis

Gene inactivationOverexpression

Page 45: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Whole genome studies

Tiling assays

Page 46: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Working with proteins• Separating proteins• Analyzing proteins and their interactions

Page 47: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Separating proteins on polyacrylamide gels

Page 48: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Immunoblot (Western blot)

Page 49: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Proteins can be sequenced

Page 50: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Complex mixtures of proteins can be analyzed by mass spectrometry

Page 51: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Liquid chromatography is used to separatepeptides before mass spectrometry

Page 52: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Mass spectrum

Page 53: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Mass spectra are compared to theoretical values

Page 54: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Figure 6.11 Genomes 3 (© Garland Science 2007)

Mouse liver proteins

Page 55: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Figure 6.20a Genomes 3 (© Garland Science 2007)

Protein interaction map of yeast

Page 56: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Nucleic acid protein interactions

Electrophoretic mobilityshift assay (EMSA)

Page 57: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Nuclease protection footprinting

Page 58: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

In vitro selectionassay

Page 59: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Chromatin immuno-precipitation (ChIP)

Page 60: Techniques of Molecular Biology. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying

Chromosome conformation capture (3C assay)