Unformatted text preview: MCB 121 2010 Dr. Ted Powers Lecture 3 Bioinformatics Gene cloning approaches (cont.) More Molecular Biology Tools Reading: Watson: pp. 750-757; 768-769 Problem Set 3
1 Last time: Constructed plasmid-based yeast genomic DNA library Isolated candidate plasmid that complemented gal mutant (phenotypic complementation) Identified Open Reading Frame in plasmid Translation product of ORF from complementing plasmid
MTKSHSEEVIVPEFNSSAKELPRPLAEKCPSIIKKFISAYDAKPDFVARSPGRVNLIGEH IDYCDFSVLPLAIDFDMLCAVKVLNEKNPSITLINADPKFAQRKFDLPLDGSYVTIDPSV SDWSNYFKCGLHVAHSFLKKLAPERFASAPLAGLQVFCEGDVPTGSGLSSSAAFICAVAL AVVKANMGPGYHMSKQNLMRITVVAEHYVGVNNGGMDQAASVCGEEDHALYVEFKPQLKA TPFKFPQLKNHEISFVIANTLVVSNKFETAPTNYNLRVVEVTTAANVLAATYGVVLLSGK EGSSTNKGNLRDFMNVYYARYHNISTPWNGDIESGIERLTKMLVLVEESLANKKQGFSVD DVAQSLNCSREEFTRDYLTTSPVRFQVLKLYQRAKHVYSESLRVLKAVKLMTTASFTADE DFFKQFGALMNESQASCDKLYECSCPEIDKICSIALSNGSYGSRLTGAGWGGCTVHLVPG GPNGNIEKVKEALANEFYKVKYPKITDAELENAIIVSKPALGSCLYEL
2 Two important web based resources: National Center for Biotechnology Information (NCBI) Saccharomyces Genome Database (SGD) 3 http://www.ncbi.nlm.nih.gov/ 4 5 6 7 Score E Sequences producing significant alignments: gi|6319494|ref|NP_009576.1| (NC_001134) Haploid specific pr... gi|171567|gb|AAA34631.1| (K01609) gal1 [Saccharomyces pasto... gi|6320212|ref|NP_010292.1| (NC_001136) Involved in galacto... gi|120899|sp|P09608|GAL1_KLULA Galactokinase (Galactose kin... gi|19113651|ref|NP_596859.1| (NC_003423) putative galactoki... gi|2494674|sp|P56091|GAL1_CANAL Galactokinase (Galactose ki... gi|6016092|sp|O42821|GAL1_CANPA Galactokinase (Galactose ki... gi|171556|gb|AAA34625.1| (M21615) galactokinase-3 [Saccharo... gi|20911655|ref|XP_130467.1| (XM_130467) RIKEN cDNA 2810017... gi|4503897|ref|NP_002035.1| (NM_002044) galactokinase 2 [Ho... gi|11259217|pir||T51592 galactokinase (EC 126.96.36.199) [validat... gi|15230749|ref|NP_187310.1| (NM_111534) galactose kinase; ... gi|6537164|gb|AAF15552.1| (AF152851) galactokinase GAL1 [Ar... gi|2292917|emb|CAA68163.1| (X99851) galactokinase [Arabidop... gi|21355577|ref|NP_648276.1| (NM_140019) CG5288 gene produc... gi|21298218|gb|EAA10363.1| (AAAB01008948) agCP2064 [Anophel... gi|21298202|gb|EAA10347.1| (AAAB01008948) agCP2077 [Anophel... gi|7295009|gb|AAF50337.1| (AE003553) CG5288 gene product [a... gi|2818|emb|CAA30089.1| (X07039) kinase (GAL 1) [Kluyveromy... gi|21291064|gb|EAA03209.1| (AAAB01008035) ebiP2014 [Anophel... gi|17508305|ref|NP_490909.1| (NM_058508) galactokinase [Cae... gi|3913721|sp|P56599|GAL1_CANMA Galactokinase (Galactose ki... gi|7505871|pir||T15285 hypothetical protein M01D7.4 - Caeno... gi|11041513|dbj|BAB17288.1| (AB050520) hypothetical protein... gi|21291063|gb|EAA03208.1| (AAAB01008035) agCP14520 [Anophe... gi|20808332|ref|NP_623503.1| (NC_003869) Galactokinase [The... gi|16764138|ref|NP_459753.1| (NC_003197) galactokinase [Sal... gi|16759699|ref|NP_455316.1| (NC_003198) galactokinase [Sal... gi|12003984|gb|AAG43832.1|AF213513_1 (AF213513) galactokina... gi|20913709|ref|XP_126664.1| (XM_126664) galactokinase [Mus... gi|8650486|gb|AAF78226.1|AF246459_1 (AF246459) galactokinas... gi|8393453|ref|NP_058601.1| (NM_016905) galactokinase [Mus ... gi|16121434|ref|NP_404747.1| (NC_003143) galactokinase [Yer... gi|21295425|gb|EAA07570.1| (AAAB01008859) agCP1294 [Anophel... gi|208230|gb|AAA72509.1| (K03394) galK [unidentified clonin... gi|12847009|dbj|BAB27400.1| (AK011103) data source:MGD, sou... gi|15830039|ref|NP_308812.1| (NC_002695) galactokinase [Esc... gi|21221574|ref|NP_627353.1| (NC_003888) galactokinase [Str... gi|2501753|gb|AAC48872.1| (U66885) galactokinase [bacteriop... gi|4503895|ref|NP_000145.1| (NM_000154) galactokinase 1 [Ho... gi|15800466|ref|NP_286478.1| (NC_002655) galactokinase [Esc... gi|3603423|gb|AAC35849.1| (AF084935) galactokinase [Homo sa... gi|6831546|sp|O85253|GAL1_THENE Galactokinase (Galactose ki... gi|96753|pir||C37760 galactokinase (EC 188.8.131.52) - Salmonell... gi|12845710|dbj|BAB26864.1| (AK010340) data source:MGD, sou... (bits) Value 1083 990 825 594 444 411 409 399 247 246 244 243 242 238 208 202 200 196 186 164 163 159 141 121 97 87 87 87 86 86 85 85 84 84 83 83 83 83 82 82 82 81 81 81 80 0.0 0.0 0.0 e-168 e-123 e-114 e-113 e-110 2e-64 5e-64 3e-63 3e-63 7e-63 2e-61 2e-52 6e-51 5e-50 4e-49 6e-46 2e-39 5e-39 9e-38 2e-32 2e-26 4e-19 5e-16 6e-16 6e-16 9e-16 1e-15 2e-15 2e-15 3e-15 3e-15 8e-15 8e-15 8e-15 1e-14 1e-14 1e-14 2e-14 3e-14 3e-14 4e-14 6e-14 8 9 10 11 12 13 http://www.yeastgenome.org/ 14 15 Yeast gene nomenclature GAL1 = YBR020W
Y = Yeast B = Chromosome II R = Gene is to the right of the centromere 020 = 20th gene from the centromere W = gene encoded on the Watson strand 16 Yeast gene nomenclature (cont.) Note: GAL1 = YBR020W GAL7 = YBR018C
17 So- the results are encouraging:
The gene you have cloned is involved in galactose metabolism But- how do you know this is the gene is mutated in your original strain? Many useful approaches, but we’ll consider just one: “Linkage Analysis” with an auxotrophic marker Takes advantage of the fact that homologous recombination is very active in yeast 18 Linkage analysis to confirm identity of the mutant gene Steps: Knock out candidate gene with a marker (e.g. HIS3) in a haploid cell Cross with mutant to form diploid Sporulate and test meiotic products from several spores 19 Linkage analysis to confirm identity of the mutant gene What would you predict? 1. If knock out and original mutant are the same gene? 2. If knock out is in a different gene?
20 Linkage analysis to confirm identity of the mutant gene Steps: Knock out candidate gene with a marker (e.g. HIS3) in a haploid cell Q: How to do this? A: Use PCR 21 22 REVIEW:
Mechanism of Polymerase Chain Reaction (PCR) Understanding requires grasping the fundamental structure of DNA: (1) Anti-parallel double stranded (Duplex) DNA (2) Complementary Base pairs 5’-GATCTAACCGTAC-3’ 3’-CTAGATTGGCATG-5’
23 Polymerase Chain Reaction
steps: 1. Denaturation (90°C) 2. Annealing (55°C) 3. Extension (70°C) Key: Thermal stabile DNA polymerase T. aquaticas (Taq) completion of 1 round 24 3 rounds of PCR are required to produce the desired product 25 Linkage analysis to confirm identity of the mutant gene Steps: Knock out candidate gene with a marker (e.g. HIS3) in a haploid cell Cross with mutant to form diploid Sporulate and test meiotic products from several spores What should the results be? 26 Linkage analysis to confirm identity of the mutant gene What would you predict? 1. If knock out and original mutant are the same gene? 2. If knock out is in a different gene?
27 Methods for isolating (cloning) genes 1. Phenotypic complementation (already discussed) 2. Hybridization-based methods 3. Expression cloning Important issue: choice of library Genomic versus cDNA 28 Genomic versus cDNA libraries Genes that are represented:
cDNA: 1. Only genes expressed in organism/cells where mRNA is isolated 2. Frequency in the library corresponds to relative abundance of transcript in mRNA population Genomic: includes all genes, whether they are expressed or not. 29 Genomic versus cDNA libraries (cont.) Sequences that are represented:
cDNA: Only sequences in mature (processed) mRNA Genomic: Includes all sequences, including promoters, other regulatory sequences, introns, and intergenic regions Note: because genes of higher eukaryotes (e.g. humans) contain many large introns, cDNA libraries are preferable for obtaining full-length ORFs. This is what we will focus on today.
30 How to make a cDNA library: key step was discovery of retroviral reverse transcriptase 31 Choice of cloning vectors and restriction site important Note: ends are now complementary to cDNA
32 Putting the pieces together: 33 How to find your gene of interest in a cDNA library? Method 1: Searching by DNA hybridization Used when: (1) The gene has been cloned and sequenced in another organism (2) Some portion of the mRNA sequence is known (3) You have a fragment of the gene in hand (e.g. by “gene walking” experiments) (4) Some of the protein sequence is known --> all of these can be used to generate a DNA probe to search the library
34 Let’s look at the 4th example: Some protein sequence is known: Trp Met Phe Lys Asn Glu Protein TGG ATG TTC AAA AAC GAA Potential TTT AAG AAT GAG gene sequence
•Use predicted gene sequence to create “oligonucleotide probes” Note, because of degeneracy of the genetic code, more than one sequence must be constructed to find the exact match! •For the example listed: 16 different “18-mers” are required to cover all possible sequences in the gene encoding this protein 35 Using the oligonucleotide probes to screen a cDNA library 36 Problem with use of degenerate single stranded oligonucleotide probes Require relatively low hybridization conditions •stringency/specificity an issue •difficulty in competing with double stranded target DNA •lead to many false-positive results Solution: If two peptide sequences are known, try and create a larger double stranded probe How? PCR and cDNA 37 How would you do this?
1. obtain peptide sequences 2. design primers: • will be degenerate • peptide 2-specific primer has complementary sequence 3. Conduct PCR rxn using cDNA as template 4. Label probe and use for hybridization with 38 cDNA library as before How to find your gene of interest in a cDNA library? Method 2: Expression cloning Used when: (1) You have purified the protein that is encoded by the gene of interest & (2) You have raised an antibody against the protein 39 Specialized plasmid necessary for expression cloning Required elements 1. inducible promoter 2. ribosomal binding site 3. multiple cloning site 4. transcription terminator alsoantibiotic resistance origin of replication 40 Outline of Expression Cloning Important caveat: protein must be recognized by the antibody when expressed in E. coli 41 Review Additional Molecular Tools Restriction Enzyme Analysis of DNA (“restriction digests”) Agarose gel: Large pore size Ideal for separation of DS DNA Electrophoresis: DNA runs toward anode Other common gel material: Polyacrylamide 42 Review Additional Molecular Tools (cont.) Southern blot analysis (named after Ed Southern)
Steps: 1. Restriction Digest & Agarose Gel 2. Transfer to membrane (nylon) 3. Probe with radio-labeled DNA or RNA 4. Detect with x-ray film/phosphoimager* *machine that detects radioactivity (more sensitive and quantitative than film) All DNA fragments in the gel can be visualized with a DNA binding dye (e.g. Ethidium Bromide) 43 Review Additional Molecular Tools (cont.)
Name of blot Southern Northern Western What’s on Gel DNA RNA Protein Gel material Agarose Agarose or PA** SDS-PA SDS-PA SDS-PA SDS-PA Probe used DNA/RNA DNA Antibody RNA DNA Protein Northwestern* Protein Southwestern* Protein Far-western Protein *variations used to detect protein-nucleic acid interactions **PA = polyacrylamide
44 Overview of Western Blotting Procedure 1 2 1 2 1 2 1 2 SDS-PAGE (polyacrylamide electrophoresis) Transfer Protein to Nitrocellulose membrane (called a “Blot”) Incubate With specific Antibody (Ab) Detect Antibody to Reveal protein Of interest 45 Put it together: can you interpret the data? 46 ...
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This note was uploaded on 09/23/2010 for the course NPB 8746546 taught by Professor Goldberg during the Spring '10 term at UC Davis.
- Spring '10