sequencing

Examples of sequencing in the following topics:

• DNA Sequencing Based on Sanger Dideoxynucleotides

  • Sanger sequencing, also known as chain-termination sequencing, refers to a method of DNA sequencing developed by Frederick Sanger in 1977.
  • More recently, dye-terminator sequencing has been developed.
  • Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day.
  • Automation has lead to the sequencing of entire genomes.
  • Different types of Sanger sequencing, all of which depend on the sequence being stopped by a terminating dideoxynucleotide (black bars).

• DNA Sequencing of Insertion Sites

  • An insertion sequence (also known as an IS, an insertion sequence element, or an IS element) is a short DNA sequence that acts as a simple transposable element.
  • The coding region in an insertion sequence is usually flanked by inverted repeats.
  • Although insertion sequences are usually discussed in the context of prokaryotic genomes, certain eukaryotic DNA sequences belonging to the family of Tc1/mariner transposable elements may be considered to be insertion sequences.
  • A complex transposon does not rely on flanking insertion sequences for resolvase.
  • It involves cutting genomic DNAs with a restriction enzyme, ligating vectorettes to the ends, and amplifying the flanking sequences of a known sequence using primers derived from the known sequence along with a vectorette primer.

• Detecting Uncultured Microorganisms

  • These advances have allowed the adaptation of shotgun sequencing to metagenomic samples .
  • The approach, used to sequence many cultured microorganisms and the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence.
  • Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples.
  • This was further followed by high-throughput sequencing which did the same process as the shotgun sequencing but at a much bigger scale in terms of the amount of DNA that could sequenced from one sample.
  • (A) sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds

• Metagenomics

  • Conventional sequencing begins with a culture of identical cells as a source of DNA.
  • The approach, used to sequence many cultured microorganisms and the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence.
  • These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; 454 pyrosequencing typically produces ~400 bp reads, Illumina and SOLiD produce 25-75 bp reads.
  • However, this limitation is compensated for by the much larger number of sequence reads.
  • An additional advantage to short read sequencing is that this technique does not require cloning the DNA before sequencing, removing one of the main biases in environmental sampling.

• Slipped-Strand Mispairing

  • Slipped strand mispairing (SSM) is a process that produces mispairing of short repeat sequences during DNA synthesis.
  • The short repeat sequences are 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences.
  • The overlapping promoter regions have repeats of the dinucleotide TA in the -10 and -35 sequences.
  • The second way that SSM induces transcriptional regulation is by changing the short repeat sequences located outside the promoter.
  • Black boxes are short sequence repeats.

• Homologs, Orthologs, and Paralogs

  • The terms "percent homology" and "sequence similarity" are often used interchangeably.
  • As with anatomical structures, high sequence similarity might occur because of convergent evolution, or, as with shorter sequences, because of chance.
  • Such sequences are similar, but not homologous.
  • Sequence regions that are homologous are also called conserved.
  • Paralogous sequences provide useful insight into the way genomes evolve.

• Mutation

  • Mutations are accidental changes in a genomic sequence of DNA; this includes the DNA sequence of a cell's genome or the DNA or RNA sequence.
  • In molecular biology and genetics, mutations are accidental changes in a genomic sequence of DNA: the DNA sequence of a cell's genome or the DNA or RNA sequence in some viruses.
  • These random sequences can be defined as sudden and spontaneous changes in the cell.
  • In general, this form of mutagenesis requires that the wild type gene sequence be known.

• Vectors for Genomic Cloning and Sequencing

  • The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
  • Vectors called expression vectors (expression constructs) are specifically for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene.
  • Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell .
  • These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible.
  • However, because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection.

• Annotating Genomes

  • Genome annotation is the identification and understanding of the genetic elements of a sequenced genome.
  • The genome sequence of an organism includes the collective DNA sequences of each chromosome in the organism.
  • For a bacterium containing a single chromosome, a genome project will aim to map the sequence of that chromosome.
  • Once a genome is sequenced, it needs to be annotated to make sense of it.
  • Genome annotation is the process of attaching biological information to sequences.

• Identification of Microbes Based on Molecular Genetics

  • A nucleic acid sequence belonging to a putative pathogen should be present in most cases of an infectious disease.
  • Fewer, or no, copies of the pathogen-associated nucleic acid sequences should occur in hosts or tissues without disease.
  • When sequence detection predates disease, or the sequence copy number correlates with severity of disease or pathology, the sequence-disease association is more likely to be a causal relationship.
  • Tissue-sequence correlates should be sought at the cellular level.
  • These sequence-based forms of evidence for microbial causation should be reproducible.