Motivation From Protein to Gene

Transcription

1 MOLECULAR BIOLOGY Topic B Recombinant DNA -principles and tools Construct a library - what for, how Major techniques +principles Bioinformatics - in brief Chapter 7 (MCB) 1 Motivation From Protein to Gene Isolate protein (in diseased blood) Determine few aa in the sequence Synthesize oligonucleotide HOW??. Isolate the gene Sequence the gene 2 1

2 Motivation From Gene to Protein Isolate genomic clone (some defect?) Isolate relevant RNA Sequence it Define the aa Comparative, search in DB Cloning in expression vector Protein production, biochemical function 3 Additional information Bioinformatics tools and methods DB search Comparative proteomics & genomics Not in this course 4 2

3 7.1 DNA cloning with plasmid vectors Recombinant DNA technology depends on the ability to produce large numbers of identical DNA molecules (clones) DNA fragment of interest is inserted into a vector DNA molecule. When a DNA in vector is introduced into a host cell, large numbers of the fragment are reproduced along with the vector Two common vectors are E. coli plasmid vectors and bacteriophage λ vectors 7.1 Plasmids are extrachromosomal selfreplicating DNA molecules Figure 7-1 3

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6 7.1 Plasmid cloning permits isolation of DNA fragments from complex mixtures Figure Restriction enzymes cut DNA molecules at specific sequences Figure 7-5a 6

7 7.1 Selected restriction enzymes Site for cutting Length of recognition site Blunt/sticky Modification sensitivity Practically - price, stability, false cut 7.1 Restriction enzymes cut DNA molecules at specific sequences Figure 7-5b 7

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9 7.1 Polylinkers facilitate insertion of restriction fragments into plasmid vectors Figure 7-8 9

10 7.2 Constructing DNA libraries with λ phage and other cloning vectors Cloning all of the genomic DNA of higher organisms into plasmid vectors is not practical. Instead vectors derived from bacteriophage are used. A collection of clones that includes all the DNA sequences of a given species is called a genomic library A genomic library can be screened for clones containing a sequence of interest 6.3 Virus/phage particles can be counted in plaque assays Figure

11 6.3 Bacterial viruses commonly used in biochemical and genetic research T phages of E. coli Temperate phages (bacteriophage λ) Small DNA phages RNA phages 7.2 The bacteriophage genome Figure

12 6.3 Bacteriophage λ undergoes either lytic replication or lysogeny following infection of E. coli ~100/cell 1000 more efficient than transformation Length up to 50 kb Figure 6-19 Lambda phage -An alternative method for genomic representation 24 12

13 7.2 Nearly complete genomic libraries of higher organisms can be prepared by λ cloning Figure

14 A library in lambda bacteriophage A larger genomic piece More effective host bacterial transfection Higher yield A genomic library (25-50 kb) A cdna library representing a tissue/condition 1. How to get all mrna in a cell? 2. How to apply cloning methods? 3. How to make mrna isolable? 27 A cdna library 1. Isolate ALL RNA (trna, mrna, rrna, srna ) 2. Purifying only mrna on polyt beads 3. Elute all mrna TTTTTTTT AAAAA AAAAA TTTTTTTT AAAAA TTTTTTTT 28 14

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18 35 Recombinant DNA technologies DETECTION METHODS Sequencing reaction (tirgul) PCR and RT-PCR (tirgul) 18

19 7.3 Identifying, analyzing, and sequencing cloned DNA The most common approach to identifying a specific clone involves screening a library by hybridization with labeled (radioactively) DNA or RNA probes. Library - A global collection of genomic region, transcribed sequences, proteins, peptides etc 7.3 The membrane-hybridization assay Melt Double stranded DNA DNA binds to filter Single-stranded DNA Filter Incubate with labeled DNA Wash away labeled DNA that did not hybridize to DAN bound to filter Hybridized complemetary DNAs Perform autoradiography Figure

20 7.1 Small DNA molecules can be chemically synthesized Synthetic DNA is useful for: generating polylinker sequences sequencing DNA isolating clones of interest creating site-specific mutations Oligonucleotide for PCR DNA chip technology. Method: 3 to 5 direction systematic exposure of reactive sites 7.3 Oligonucleotide probes design Probes - Unique (20 mer = ) For protein - 7 aa Degenerative probes Figure

21 7.3 Oligonucleotide probes are designed based on partial protein sequences Labeled - 5 polynucleotide kinase Figure

22 Probes may be Synthetic poly-nucleotide mrna (or any genomic/ genetic string) Antibodies Peptides Drug Specific clones can be identified based on properties of the encoded proteins Figure

23 Typical Questions in Molecular research 1. Homologue gene from yeast to man? 2. An antibody in serun of diseased condition? 3. Cancer cell -Any new gene is expressed? 4. Treated with drug -which transcript is suppressed? 5. Parenthood, criminal evidence - profile? 6. And many, many more. 7.3 Gel electrophoresis resolves DNA/protein fragments of different size Figure

24 7.3 Visualization of restriction fragments separated by gel electrophoresis Figure Southern blotting detects specific DNA fragments Figure

25 7.5 Northern blotting detects specific mrnas Figure 7-33 Western Blot - your protein Separation on gel a protein mixture Blotting (solid phase matrix) Specific probe (antibodies) Detection method (enzyme, Radiolabel, luminescence ) 25

26 7.3 DNA sequencing: the Sanger method Four separate polymerization reactions are performed Figure 7-29a 26

27 7.3 DNA sequencing: the Sanger (dideoxy) method Figure 7-29b,c 27

28 Few words on ESTs Large set of expressed cdna partially sequenced nt Over 3 million public In silico cloning Very important source for finding new genes, alternative spliced etc. Figure

29 Few words on PCR Alternative technology to classical cloning Method to recover minute amounts of DNA (crime scene) Method to detect Alternative splicing Method to introduce mutations Many more. Figure

30 Producing high levels of proteins from cloned cdnas Many proteins are normally expressed at very low concentrations within cells, which makes isolation of sufficient amounts for analysis difficult To overcome this problem, DNA expression vectors can be used to produce large amounts of full length proteins 30

31 7.6 E. coli expression systems can produce full-length proteins Figure Even larger amounts of a desired protein can be expressed with a two-step system Figure

32 7.4 Bioinformatics Bioinformatics is the rapidly developing area of computer science devoted to collecting, organizing, and analyzing DNA and protein sequences Using searches based on homologous sequences, stored sequences suggest functions of newly identified genes and proteins New technologies New opportunities to understand the molecular level of life The role of Bioinformatics in the Human Genome Project 32

33 7.4 The C. elegans genome encodes numerous proteins specific to multicellular organisms Analyzing complex mixtures A cellular snapshot Detect the presence and the amounts of complementary nucleic acids in complex mixtures including total cellular RNA 33

34 7.8 DNA microarrays: analyzing genome-wide expression DNA microarrays consist of thousands of individual gene sequences bound to closely spaced regions on the surface of a glass microscope slide DNA microarrays allow the simultaneous analysis of the expression of thousands of genes The combination of DNA microarray technology with genome sequencing projects enables scientists to analyze the complete transcriptional program of an organism during specific physiological response or developmental processes 34

35 7.8 A yeast genome microarray Figure

36 DNA Chip technology The yeast genome -a snapshot Clustering of expression data the cell program 36

37 7.8 Changes in yeast gene expression as cells deplete glucose from the growth media Figure 7-40a DeRisi: Coordinated regulation of functionally-related genes 37

38 DeRisi: Expression Time Course DNA Chip technology Currently, up to 20,000 DNA samples, or clones, can be arrayed on each microarray. DNA samples can include genes with known functions DNA samples can also include gene fragments (ESTs) whose function is unknown. 38

39 DNA CHIP analysis in determine the profile of a specific type of cancer Microarray Applications Detect expression of thousands of genes Many applications Identification of complex genetic diseases Drug discovery and toxicology Mutation and polymorphism (SNP) detection Pathogen analysis Detect patterns of gene expression between tissues or disease states 39

40 7.3 Additional material For your own fun Technologies 7.3 Pulsed-field gel electrophoresis separates large DNA molecules Chromosoe Separation Chromosome specific libraries Figure

41 7.5 Specific RNAs can be quantitated and mapped on DNA by nuclease protection Figure 7-34a,b 7.5 Transcription start sites can be mapped by S1 protection and primer extension Figure

42 RNA association Eukaryotic tissue 10,000-15,000 mrna ~5 copies ~4,000 copies ~100,000 copies abundance 84 42

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