Transcript lecture-3-techniques-of-molecular-biologyx

Techniques of Molecular Biology
Basic molecular biology techniques
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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
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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
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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
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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
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Isolating nucleic acids
Cutting DNA into fragments
Ligating DNA fragments
Amplifying DNA fragments
Hybridization techniques
Genomics
• Sequencing genomes
Sequencing techniques
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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
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dideoxysequencing
pyrosequencing
Base
Base
Sequencing techniques
2’-3’-dideoxynucleotide
2’ deoxynucleotide
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dideoxysequencing
Base
Base
ddCTP
Sequencing techniques
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ddTTP
ddGTP
dideoxysequencing
polyacrylamide gel
electrophoresis
≈ 800 nucleotides
can be sequenced
in one run
Sequencing techniques
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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
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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
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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