DNA Sequence Analysis

  1. The Maxam-Gilbert method of nucleotide sequence determination

    relies on enzymatic modification of DNA to generate a nested set of end-labeled derivatives.

    is based on preferential, base-specific methylation followed by chemical cleavage to generate a nested set of end- labeled derivatives.

    is dependent on the differential sensitivity of the normal DNA nucleotides to chemical cleavage by piperidine.

    is inherently safer and more reproducible than the dideoxy or Sanger method of nucleotide sequence determination.

  2. The Sanger or dideoxy method of nucleotide sequence determination

    relies on sequence-specific termination of an enzymatically synthesized DNA chain.

    requires some knowledge about the nucleotide sequence of the region to be characterized.

    is much less common the the Maxam-Gilbert method of sequence analysis.

    is inherently complicated by the requirement for a binding site for a sequencing primer.

  3. The sequencing of a typical 5000 base pair cloned DNA fragment can be accomplished by

    merely initiating the sequencing reaction at one end of the cloned fragment and reading the sequence through the desired entire length.

    sequencing as far as possible, then chemically synthesizing a new sequencing primer and continuing with a new sequence reaction.

    sequencing of overlapping, subcloned restriction fragments derived from the larger fragment.

    sequencing of a set of terminal deletions of the large fragments to generate an overlapping set of nested sequences.

  4. Types of polynucleotide that can be directly sequenced by the enzymatic approach include


    single-stranded DNA.

    double-stranded DNA cloned in a vector.

    PCR-amplified DNA.

  5. Nucleotide sequence analysis of a cDNA molecule isolated from a tissue-specific cDNA library generally does not provide information about

    the amino acid sequence of the encoded gene product.

    the site of transcription initiation.

    splicing and processing sites within the mRNA molecule.

    regulatory sequences that influence mRNA expression levels.

    ©1999 Attotron Biosensor Corporation
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