Genomes can have known sequences {DNA sequencing}. H. influenzae has 1.8 million bases and 800 genes. E. coli has 4.5 million bases and 2000 genes. Saccharomyces yeast has 12 million bases and 6000 genes. P. falciparum has 30 million bases and 6500 genes. Caenorhabditis elegans roundworm nematode has 100 million bases and 10000 genes. Arabidopsis thaliana weed has 120 million bases and 20000 genes. Drosophila melanogaster fruitfly has 165 million bases. Fugu rubripes is zebrafish. Mus musculus mouse has 3000 million bases. Humans have 3500 million bases and 30000 genes.
methods
Sequencing {plus-minus method} can elongate DNA sequences using DNA polymerase.
Sequencing {Maxam-Gilbert sequencing method} can chemically degrade labeled, short DNA chains at G, C, A and G, and T and C to make fragments and then electrophorese all four, separately or together, to separate by size.
Sequencing {Sanger sequencing method} can elongate DNA chains and randomly terminate them with nucleotides that cannot bind to next ribose, using A, G, T, and C 2',3'-dideoxynucleoside triphosphates, and then electrophoreses all four, separately or together, to separate by size.
Mixing differently labeled clones {multiplex DNA sequencing} allows easier processing. Labels identify clones.
DNA-fragment ends can have fluorescent dyes, which respond to laser light. Dye terminator attaches to nucleotide 3' ends. Dye primer attaches to 5' ends. Longer fragments have higher signals. Fragments separate by electrophoresis in gels {horizontal ultrathin gel electrophoresis} (HUGE) or capillary tubes {capillary gel electrophoresis}. Because charges are equal, fragments leave gel or tube by size.
Scanning tunneling electron microscopes can sequence DNA strands by wand distance needed to maintain constant voltage. They can sequence 40000-base DNA fragments one base at a time. Exonuclease removes end nucleotide. Shining laser light and recording with photomultiplier reads nucleotide type.
Gas or liquid mixtures can separate using gas chromatography, liquid chromatography, supercritical fluid chromatography, or capillary electrophoresis.
Computers overlap sequence information about DNA fragments {contig, sequencing} to build longer sequences, to map large fragments and then chromosomes.
Cluster hierarchies can look like tree diagrams {dendrogram, sequencing}.
If many DNA fragments insert into many bacteria, plate colonies can account for all DNA fragments {DNA library} from foreign organisms.
DNA analyses include comparing DNA sequences. Sequences of same gene from different organisms can align somewhat {homology, DNA}| {homologous sequences}.
Probes hybridize with clone and fragment regions. Overlapping DNA fragments hybridize to same probe. 20-base oligonucleotides can be probes to find overlapping DNA fragments. For clone ends, random sequences can be probes {sequence-tagged connector} (STC). Probes can have minor-groove binder to enhance exact hybridization, allowing shorter probes.
Processing identifies unique 200-base to 500-base sequences, with unique primers, from known locations {sequence-tagged site} (STS). Perhaps, clones share STS.
After DNA fragments exit capillary, plot {electropherogram} shows relative dye concentration versus time expressed as frame number. In electropherograms, small peaks {pull-up peak} can appear under main dye peaks. Incorrect dyes, dye contamination, or capillary or fluid-property changes after spectral calibration can cause pull-up peaks.
In direct methods, mRNA-AAAAA + reverse transcriptase + Oligo-dT Primer + dNTPs + Cy3 and Cy5 or SymJAZ dye-dNTP -> Dye-cDNA {DNA labeling}.
In random priming methods, mRNA + reverse transcriptase + T7-T20-24 + MuLV -> DNA/RNA + hydrolysis -> cDNA first strand + Bst DNA polymerase + ligase + pN8-9 -> T7-ds cDNA + dye-UTP + T7 RNA polymerase + IVT -> labeled cRNA.
In RNase H methods, mRNA + reverse transcriptase -> DNA/RNA + RNase H -> DNA/RNA + DNA polymerase + ligase -> T7-ds cDNA + dye-UTP + T7 RNA polymerase + IVT -> labeled cRNA.
purposes
DNA labeling can measure labeled-cRNA dye incorporation, reverse-transcriptase conversion, fluorescence-specific activity, minimum RNA, maximum RNA, IVT amplification, total amplification, and length.
controls
Control reagents aid spot finding, image analysis, and signal quantification. Array probes monitor spotting, labeling, hybridization, printing, attachment, and features. Labeling controls monitor enzyme activity, target stability, and dye incorporation during labeling protocols. Hybridization controls monitor mixing, stringency, and washing during array-hybridization protocol. Printing and attachment controls monitor array manufacturing, probe attachment, and lot-to-lot variability. Feature controls normalize signal variability.
DNA analyses include finding sequences longer than cloned DNAs by looking for their overlaps. First clone can screen whole-genome clone library for overlapping sequences. If overlap, second clone can screen, and so on, to build longer and longer sequences {chromosome walking}.
DNA sequencing can use modified shotgun methods {polony sequencing}.
DNA-fragment mixtures can separate fragments by lengths {fragment analysis}.
Electrophoresis gels have band sequences {lane}, starting from top sample bands.
Electrophoresis separates eluted DNA fragments, to compare peak separations, spectral separations, and spatial separations {fragment separation}.
Compounds less soluble in stationary phase elute faster {retention time, elution}.
DNA analyses can separate cell RNAs or mRNAs on gels and transfer gel-band contents to filters, where RNAs can hybridize to known RNA or cDNA sequences {Northern blotting}. Northern blotting compares mRNAs to cDNAs to study gene expression.
DNA analyses can separate DNA fragments on gels and transfer gel-band contents to filters, where DNA fragments can hybridize to known DNA sequences {Southern blotting}. Southern blotting can detect gene rearrangements that make antibodies and T-cell receptors. It can detect disease-caused gene rearrangements and deletions. It can detect related genes in organisms and homologous genes from different species. It can detect mRNA-splicing-caused intron removal and exon use. It can detect mRNA splicing to make alternative proteins. It can detect nested genes.
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Date Modified: 2022.0225