DNA SEQUENCE ANALYSIS DNA SEQUENCES Different methods Kinds of DNA that can be sequenced Obtaining good quality

sequences DNA sequencing services

Different methods Automated fluorescence DNA sequence methods Next generation Sequencing

AUTOMATED FLUORESCENCE DNA SEQUENCE METHODS Variation of the Sanger chain-termiantion protocol Template + primer +dNTPs + ddNTPs +Polymerase enzyme +buffer Cycle sequencing reaction : denaturation (95-96%), annealing (50-55%) and extension (60-70%) 2 general categories of sequencing chemistry approaches: Dye-primer and Dye-terminator chemistries
(see http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/dna-sequencingfragment-analysis/overview-of-dna-sequencing/sequencing-chemistries.html

*4 steps in Dye-primer each of the 4 sequence reaction will contain either a blue, green, yellow or red labeled primer. Color corresponds to ACTG. ddNTPs are present in each reaction mixture and randomly terminate DNA synthesis, creating DNA fragments of Different lengths dyes are attached to the ddNTPs. Requires only one reaction tube per sample instead of 4 DNA template. Unlabeled primer, buffer, 4dNTPs, 4 fluorescent labeled ddNTPs, AmpliTaq were added to the reaction tube. Fluorescent fragments are generated. About the Kit...

New formulation, the Big Dye Terminator v3.1 cycle sequence kit has a new formulation that delivers -Increase robustness -More even peak heights -Longer read lengths

Levels of DNA that can be sequenced DNA Type Plasmid Cosmid PCR product Single stranded phage BAC/PAC Concentration (ng/L) 200-250 200-250 10-20 100-150 300-400 (best to sequence) Total used in the reaction 500-700 ng 500-700 ng 10 ng/100 bp 300 ng 700-900 ng

Obtaining good quality Sequence Most important factor Template purity and template concentration TEMPLATE PURITY Contaminant RNA PCG NaOAc Ethanol Phenol inhibit enzyme so panget tempate CsCl EDTA PCR Product Purification Amicon Centricon Centrifugal Filter device (millipore) High pure PCR Product Purification Kit (Roche) PRINCIPLE: Amount tolerated in sequence reaction 1 g 0.3% 5-10mM 1.25% 0% 5mM 0.25mM

TEMPLATE CONCENTRATION Quantitation by: Spec, AGE, and Fluorometry SPEC Least accurate, but most convenient NanoDrop -UV/Vis analyses of 1l sample -No need for cuvettes and capillaries -Nucleic acid absorbs strongly at 260nm -Protein contamination detected at 280nm -DNA quality and purity : A260/A280 = 1.8-2.0 -DNA concentration: 50g/ml per unit A260 -RNA concentration : 40g/ml per unit A260 AGE Can visualize contaminating DNA or RNA but not proteinor other chemicals Needs experience and patience FLUOROMETRY

 Fluorometer Most-accurate Not affected by common contaminants in DNA preparation Can detect DNA as low as 5ng/l DNA Sequencing services Hongkong Korea Japan Malaysia Singapore 1st BASE DNA $5/sample, for 20 or more sample, they pay for courier service (Asiagel) 25-50 ng/l concentration of vacuum dried DNA; include primers (10l of primer pere 5 sample rxn) Results available within 4-5 days upon receipt of sample SEQUENCE ANALYSIS Interpreting genes Troubleshooting your data Gen. Analysis strategy Assembling the sequenced part of the gene Multigene sequence analysis TechDragon Macrogen Hokkaido system science 1st BASE 1st BASE

NOTE: -If 3000 bp yung need mo isequence, tas 500 bp lang yung nareread do multiple sequence analysis (to see homology/ alignment) -Sequence data at BEGINNING, MIDDLE & END BEGINNING Peaks are not well resolved, smaller; not very reliable MIDDLE where good stuff is; peaks are sharp, high & well-designed and spaced END Resolution of the peaks begin to deteriorate; broad and round in shape Factors that cannot be controlled - No sequence data CAUSE: Priming site not present; not enough DNA or RNA; inhibitory contaminant SOLUTION: double check quantitation, stock concentration calculation, dilutions, template purity and concentration, precipitation - Imaging data with weak signals

CAUSE: Not enough DNA, inhibitory contaminants, degraded DNA from nuclease, repeated freeze-thaw SOLUTION: double check quantitation, make fresh stock reagents, use high quality distilled water Multiple peaks within your sequences Location of multiple peaks (BEGINNING, MIDDLE OR END?) *BEGINNING??? CAUSE : Multiple priming sitein vector/PCR, residual primer or dNTPs SOLUTION : Template purity and preparation, redesign primers, optimization of PCR reaction *FARTHER??? CAUSE :Mixed plasmid preparation, compression SOLUTION : pick single colony (clonal), alter cycle sequencing conditions *END??? CAUSE : dye blobs; spikes; loss of resolution SOLUTION : Template purity/preparation, re-run sample Over all signal strength (above 100 or so?)

Gen. Analysis Strategy Analyze forward and reverse sequences -Visual check of chromatogram and sequences print out - Correct/resolve ambiguous bases Search Homology (use software) Final DNA sequence and translated to Amino acid sequences Assembling the sequence part of the gene If may overlaps, tignan sa chromatogram kung san sila nagooverlap Automatic SeqScape software from ABI Manually short sequence of a gene are analyzed and assembled Multiple Sequence Analysis ClustalX BioEdit MEGA Blast H.pylori CagA gene 1.) H.pylori found in stomach and implicated in gastric cancer 2.) CagA gene 3.) Full CagA gene sequence flowchart - Sequence analyses - Phylogenetic analyses H.pylori - Group 1 carcinogen in humans - Strain specific genetic diversity postulated to be involved in the organisms ability to cause diff. Disease outcomes - Indicator of geographic diff. Among strains CagA gene

CagA -

One of the several gene of the pathogenecity island (Cag PAI) Encodes for CagA proteins proteins Virulence factor of H.pylori

Chemical cause of H.pylori

Western and East Asian countries hypes - With char. Protein EPIYA Western countries East Asian countries EPIYA Type ABC ABD

RECOMBINANT DNA Creation of new combination of DNA segments that are not found in the nature Example : Bacterial DNA +Human DNA = Chimeric DNA 1955 Watson and Crick 1967 Kornberg DNA polymerase 1975 Gellert DNA Ligase 1985 Sanger and Gilbert 1991 Mullis PCR 1995 Hood & Hunkapilla-new automated DNA tech. 2001 Consortia of Human genome sequence RESTRICTION ENZYMES Bacteria have learned to restrict the possibility of attack from foreign DNA by means of restriction enzymes Cut up foreign DNA Type II & III restriction enzyme cleaves DNA chains at selected areas Type II restriction enzymes EcoRI HindIII Bq/a PstI BamHI DpnI Additional Tool (go to google books- MOLECULAR BIOLOGY OF THE CELL) - ATP, dNTPs, Linkers - Transposable elements Klenow fragments of DNA POL I: converts sticky ends to blunt ends Terminal deoxynucleotidyl transferase PLASMID: VEHICLES OF CLONING - Naturally occuring extra chromosomal DNA molecule

- Circular. DsDNA - Can be cleaved by restriction enzyme having sticking/blunt ends CLONING VECTOR - Origin of replication - Selectable marker Cloning vector must - Multiple cloning sites to insert foreign DNA - Host DNA CHOICE: DEPENDING ON INSERT SITE AND APPLICATION Plasmid - Insert size 10kb - Can be used in sub cloning Cosmid - Insert size 35-45kb - Cos site (bacteriophage lambda) provide blunt ends Phage?? - 5-20 kb - Fusion to host protein - Cdna cloning BAC - Bacterial artificial chromosome - Insert size 73-300kb - Analysis of large genome YAC - Yeast - Yeast centromere, telomere, autonomously replicative sequences - 100-1000kb - Analysis of larger genome, YAC transgenic mice MAC - Mammalian - Mammalian centromere, telomere, origin of replication - 100kb to >1Mb Transformation - Natural process of horizontal gene transfer among bacteria - Transdauction (phage DNA) - Transfection (non-bacterial host cell) Methods of Transformation - Competent cell production - Electroporation - Microinfection