Happy New Year! Welcome to BIO372 - Molecular Biology 2016 Milestones in Biology Figure 5.2a Electrophoretic mobility of DNA fragments of various sizes Pulsed field gel electrophoresis of yeast chromosomal DNA -ca. 2 MB -ca. 0.2 MB Capillary blotting of DNA fragments on membranes See article by E.M. Southern (1975) Cited 32,000 times! DNA fingerprinting with repetitive elements DNA fingerprinting of individuals hap1 Probe hap2 Probe hap1 diploid hap2 Allele-specific probes for SNP detection..AGGTCATTGATAGTACGGTAA....AGGTCATTGACAGTACGGTAA.. * GTAACTATCATGC-32P GTAACTGTCATGC-32P S1 nuclease mapping of the 5 end of a transcript. Description:Calf Intestinal Alkaline Phosphatase (CIAP) is a phosphomonoesterase that removes 3 and 5 phosphates from DNA and RNA. Applications: Dephosphorylation of 5-phosphorylated termini of vector DNA to prevent self-ligation (1). Dephosphorylation of 5 termini of nucleic acids prior to forward reaction with kinase. Source: Purified from calf intestinal mucosa. Performance and Quality Testing: Endodeoxyribonuclease, 3 exodeoxyribonuclease, and ribonuclease assays; dephosphorylation efficiency measured in a transformation assay. Unit Definition: One unit hydrolyzes 1 mol of 4-nitrophenyl phosphate in 1 min. at 37C. Unit Reaction Conditions: 1 M diethanolamine buffer, 10 mM 4-nitrophenyl phosphate, 0.25 mM MgCl 2 (pH 9.8) in 900 l for 10 min. at 37C. Contents and Storage:Calf Intestinal Alkaline Phosphatase is supplied with a vial of 10X dephosphorylation buffer [500 mM Tris-HCl (pH 8.5), 1 mM EDTA], vial of dilution buffer [50% glycerol, 25 mM Tris-HCl (pH 7.6), 1 mM MgCl 2, and 0.1 mM ZnCl 2 ]. Store Calf Intestinal Alkaline Phosphatase at - 20C. Reference(s):1. Schmidt, B. et al. (1998) Focus 20: 52. Source:www.invitrogen.com Description: Bacterial Alkaline Phosphatase (BAP) removes 3 and 5 phosphates from DNA and RNA. BAP is active at 65C for at least 1 h and is inactivated by phenol extraction. Applications: Dephosphorylation of 5-phosphorylated termini of vector DNA to prevent self-ligation. Dephosphorylation of 5 termini of nucleic acids prior to forward reaction with kinase. Source: Purified from E. coli C90. Performance and Quality Testing: Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; dephosphorylationefficiency determined. Unit Definition: One unit hydrolyzes 1 nmol of ATP in 30 min. at 37C. Unit Reaction Conditions: 10 mM Tris-HCl (pH 8.0), 1.5 mM [- 32 P]ATP, and enzyme in 50 l for 30 min. at 37C. Contents and Storage:Bacterial Alkaline Phosphatase is supplied with a vial of 10X dephosphorylation buffer [100 mM Tris-HCl (pH 8.0)]. Store at -20C. Source:www.invitrogen.com Description:T4 Polynucleotide Kinase catalyzes the transfer of the -phosphate of ATP to the 5 hydroxyl terminus of DNA or RNA.A T4 Polynucleotide Kinase Technical Bulletin is available. Applications: 5 end-labeling of DNA or RNA (1). Phosphorylation of synthetic linkers (1). Source: Purified from E. coli expressing the T4 Polynucleotide Kinase gene in a vector. Performance and Quality Testing: Ribonuclease, endodeoxyribonuclease, 3 and 5 exodeoxyribonuclease, and phosphatase assays;labeling efficiency tested. Unit Definition: One unit transfers 1 nmol of phosphate of ATP to the 5 hydroxyl termini of micrococcal nuclease-treated DNA in30 min. at 37C. Unit Reaction Conditions: 70 mM Tris-HCl (pH 7.6), 0.1 M KCl, 10 mM MgCl 2, 1 mM 2- mercaptoethanol, 0.3 mM [- 32 P]ATP,0.38 mg/ml micrococcal nuclease-treated DNA, and enzyme in 150 l for 30 min. at 37C. Contents and Storage:T4 Polynucleotide Kinase is supplied with a vial of 5X forward reaction buffer [350 mM Tris-HCl (pH 7.6), 50 mM MgCl 2, 500 mM KCl, 5 mM 2-mercaptoethanol] and a vial of 5X exchange reaction buffer [250 mM imidazole-HCl (pH 6.4), 60 mM MgCl 2, 5 mM 2- mercaptoethanol, 350 M ADP]. Store at -20C. Reference(s):1. T4 Polynucleotide Kinase Technical Bulletin. Description:S1 Nuclease is a single-strand-specific endonuclease that hydrolyzes single-stranded RNA or DNA into 5 mononucleotides. The enzyme will hydrolyze single- stranded regions in duplex DNA such as loops and gaps. S1 Nuclease is stable at 65C. Applications: Nuclease mapping techniques (1,2). Removal of single-stranded regions from double-stranded DNA (3). Exo III-ordered sequencing (4).Source: Isolated from Aspergillus oryzae. Performance and Quality Testing: Double-strand-specific deoxyribonuclease and phosphatase assays. Unit Definition: One unit hydrolyzes 1 g of denatured DNA to acid-soluble material in 1 min. at 37C. Unit Reaction Conditions: 30 mM sodium acetate (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 0.5 mg/ml heat-denatured DNA, 5% (v/v) glycerol, and enzyme in 0.5 ml for 10 min. at 37C. Contents and Storage:S1 Nuclease is supplied with a vial of 10X S1 Nuclease buffer [300 mM sodium acetate (pH 4.6), 10 mM zinc acetate, 50% (v/v) glycerol], vial of dilution buffer, vial of 3 M NaCl. Store at -20C. Reference(s):1. Berk, A.J. et al. (1977) Cell 12: Berk, A.J. (1981) Focus 3: 1.3. Gerard, G.F. (1985) Focus 7: 7.4. Hoheisel, J. et al. (1986) Nucleic Acids Res. 14: 3605. S1 mapping of the 3 end of a transcript Sanger sequencing of DNA Figure 5.19 Sanger sequencing of DNA Figure 5.20b Sequence read Primer extension Description:SuperScript Reverse Transcriptase (RT) is a DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene from which the RNase H sequence has been deleted, reducing the RNase H activity. This structural modification prevents degradation of the RNA molecule during first- strand cDNA synthesis (Figure 1). Purified from E. coli expressing the pol gene of M-MLV (4,5), mutagenized to reduce the RNase H activity (6). Unit Description:One unit incorporates 1 nmol of deoxyribonucleotide into acid-precipitable material in 10 min. at 37C using poly(A)oligo(dT) 1218 as templateprimer (7). Unit Reaction Condition:50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl 2, 1 mM DTT, 0.5 mM [ 3 H]dTTP, 0.1 mM poly(A), 0.1 mM oligo(dT) 1218, 0.1 mg/ml BSA, and enzyme in 50 l for 10 min. at 37C. Contents and Storage:SuperScript RNase H - Reverse Transcriptase is supplied with a vial of 5X first- strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl 2 ], vial of 100 mM DTT. Store at -20C. Reference(s):1. Gerard, G. (1989) Focus 11: Howland, P. et al. (1991) Mol. Brain Res. 11: Gerard, G.F. et al. (1993) in Methods in Molecular Biology, Vol 16: Enzymes of Molecular Biology (Burrell, M. Ed.), Humana Press, Totowa, N.J., p Kotewicz, M. et al. (1985) Gene 35: Kotewicz, M.L. et al. (1988) Nucleic Acids Res. 16: Houts, G. et al. (1979) J. Virol. 29: 519. Run-off transcription Nuclear run-on transcription The cat reporter gene to measure transcription Accumulation of acetylated CAM Nitrocellulose filter binding assay Gel mobility shift assay DNase footprinting Next generation DNA sequencing Illumina sequencing 454 sequencing Genome-wide mutation dynamic within a long-lived individual of Armillaria Time The fungal individual over time model 1 Present Start Population 20 m Rhizomorphs collected from 29 locations on Exe Island, Portland, Ontario, Canada. Locations surveyed by a combination of GPS and tape measure/compass. Sample of Armillaria gallica Sequence variation in ten PCR amplified segments of DNA from Hodnett and Anderson Somatic incompatibility compatible incompatible indiv. 1 indiv. 2 indiv. 1 indiv. 2 #7 #9 #14 Genome sequencing Illumina sequencing of several libraries of no. 7 (BGI) 90 MB de-novo assembly with 7502 scaffolds, low repetitive content, 20,000 genes All ten sequences used in genotyping aligned cleanly to the assembly A and B mating type genes identified Reliable framework for navigating the genome and SNP discovery Two collections re- sequenced for SNP discovery #7 #9 #14 SNP discovery Ca. 329,608 SNPs called in no 7. 66,525 SNPs in no. 14, that are homozygous in no. 7 87,695 SNPs in no. 9 that are homozygous in no. 7 Most of the SNP differences among the sequenced collections are due to false positives/negatives. Validation test was PCR & Sanger sequencing. Filtered for highest-quality scores and for zero reads of alternate allele in putative homozyotes. Efficiency of detection, about 25%. (ca. 100 candidates tested in total) Obtained Sanger sequences for candidate sites over entire collection #7 #9 #14 Spatial distribution of mutant genotypes