sequencing

Examples of sequencing in the following topics:

• DNA Sequencing Techniques

  • The Sanger sequencing method was used for the human genome sequencing project, which was finished its sequencing phase in 2003, but today both it and the Gilbert method have been largely replaced by better methods.
  • When the human genome was first sequenced using Sanger sequencing, it took several years, hundreds of labs working together, and a cost of around $100 million to sequence it to almost completion.
  • Sanger sequence can only produce several hundred nucleotides of sequence per reaction.
  • Most next-generation sequencing techniques generate even smaller blocks of sequence.
  • Most genomic sequencing projects today make use of an approach called whole genome shotgun sequencing.

• Strategies Used in Sequencing Projects

  • The strategies used for sequencing genomes include the Sanger method, shotgun sequencing, pairwise end, and next-generation sequencing.
  • All of the segments are then sequenced using the chain-sequencing method.
  • A larger sequence that is assembled from overlapping shorter sequences is called a contig.
  • This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.
  • Compare the different strategies used for whole-genome sequencing:  Sanger method, shotgun sequencing, pairwise-end sequencing, and next-generation sequencing

• Use of Whole-Genome Sequences of Model Organisms

  • The first genome to be completely sequenced was of a bacterial virus, the bacteriophage fx174 (5368 base pairs).
  • This was accomplished by Fred Sanger using shotgun sequencing.
  • Several other organelle and viral genomes were later sequenced.
  • Having entire genomes sequenced aids these research efforts.
  • The process of attaching biological information to gene sequences is called genome annotation.

• Uses of Genome Sequences

  • Genome sequences and expression can be analyzed using DNA microarrays, which can contribute to detection of disease and genetic disorders.
  • Almost one million genotypic abnormalities can be discovered using microarrays, whereas whole-genome sequencing can provide information about all six billion base pairs in the human genome.
  • Although the study of medical applications of genome sequencing is interesting, this discipline tends to dwell on abnormal gene function.
  • Genomics is still in its infancy, although someday it may become routine to use whole-genome sequencing to screen every newborn to detect genetic abnormalities.
  • It sounds great to have all the knowledge we can get from whole-genome sequencing; however, humans have a responsibility to use this knowledge wisely.

• Noncoding DNA

  • Noncoding DNA are sequences of DNA that do not encode protein sequences but can be transcribed to produce important regulatory molecules.
  • In genomics and related disciplines, noncoding DNA sequences are components of an organism's DNA that do not encode protein sequences.
  • Other noncoding sequences have likely, but as-yet undetermined, functions.
  • Some sequences may have no biological function for the organism, such as endogenous retroviruses.
  • More than 98% of the human genome does not encode protein sequences, including most sequences within introns and most intergenic DNA.

• Physical Maps and Integration with Genetic Maps

  • Physical maps display the physical distance between genes and can be constructed using cytogenetic, radiation hybrid, or sequence mapping.
  • There are three methods used to create a physical map: cytogenetic mapping, radiation hybrid mapping, and sequence mapping.
  • Sequence mapping resulted from DNA sequencing technology that allowed for the creation of detailed physical maps with distances measured in terms of the number of base pairs.
  • A genetic site used to generate a physical map with sequencing technology (a sequence-tagged site, or STS) is a unique sequence in the genome with a known exact chromosomal location.
  • An expressed sequence tag (EST) and a single sequence length polymorphism (SSLP) are common STSs.

• Initiation of Transcription in Prokaryotes

  • A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription .
  • The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently.
  • The -10 consensus sequence, called the -10 region, is TATAAT.
  • The -35 sequence, TTGACA, is recognized and bound by σ.
  • The σ subunit of prokaryotic RNA polymerase recognizes consensus sequences found in the promoter region upstream of the transcription start sight.

• Molecular Analyses and Modern Phylogenetic Trees

  • Evolutionary trees can be made by the determination of sequence information of similar genes in different organisms .
  • Sequences that are similar to each other frequently are considered to have less time to diverge, while less similar sequences have more evolutionary time to diverge.
  • The evolutionary tree is created by aligning sequences and having each branch length proportional to the amino acid differences of the sequences.
  • Furthermore, by assigning a constant mutation rate to a sequence and performing a sequence alignment, it is possible to calculate the approximate time when the sequence of interest diverged into monophyletic groups.
  • Sequence alignments can be performed on a variety of sequences.

• mRNA Processing

  • While RNA Polymerase II is still transcribing downstream of the proper end of a gene, the pre-mRNA is cleaved by an endonuclease-containing protein complex between an AAUAAA consensus sequence and a GU-rich sequence.
  • Intron sequences in mRNA do not encode functional proteins.
  • For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.
  • Spliceosomes recognize sequences at the 5' end of the intron because introns always start with the nucleotides GU and they recognize sequences at the 3' end of the intron because they always end with the nucleotides AG.
  • This cleavage is done by an endonuclease-containing protein complex that binds to an AAUAAA sequence upstream of the cleavage site and to a GU-rich sequence downstream of the cut site.

• The DNA Double Helix

  • If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG.
  • A mutation is a change in the sequence of the nitrogen bases.
  • For example, in the sequence AATTGGCC, a mutation may cause the second T to change to a G.
  • Most of the time when this happens the DNA is able to fix itself and return the original base to the sequence.