DNA Sequence Analysis

The Maxam-Gilbert method of nucleotide sequence determination
relies on enzymatic modification of DNA to generate a nested set of end-labeled derivatives.
False! This method is chemical, rather than enzymatic.
is based on preferential, base-specific methylation followed by chemical cleavage to generate a nested set of end- labeled derivatives.
Correct!
is dependent on the differential sensitivity of the normal DNA nucleotides to chemical cleavage by piperidine.
False! The bases must be methylated prior to chemical cleavage.
is inherently safer and more reproducible than the dideoxy or Sanger method of nucleotide sequence determination.
False! The volatile and hazardous reagents make this method more difficult and much less commonly applied.

The Sanger or dideoxy method of nucleotide sequence determination
relies on sequence-specific termination of an enzymatically synthesized DNA chain.
Correct! Chain termination is accomplished by the incorporation of a dideoxynucleoside triphosphate.
requires some knowledge about the nucleotide sequence of the region to be characterized.
Correct! The reaction is initiated by the annealing of a sequencing primer, which may occur to a site in either the cloning vector that carries the fragment to be sequenced or in the fragment itself.
is much less common the the Maxam-Gilbert method of sequence analysis.
False! The vast majority of routine sequence analysis to date has been performed by one of several variations of the Sanger approach.
is inherently complicated by the requirement for a binding site for a sequencing primer.
False! Virtually all routine DNA cloning vectors have binding sites for commercially available sequencing primers.

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.
False! Typical sequencing reactions have a size limitation that precludes reliable sequence determination for distances greater than a few hundred base pairs.
sequencing as far as possible, then chemically synthesizing a new sequencing primer and continuing with a new sequence reaction.
Correct! It may require several successive rounds of sequence reactions, but this allows the compilation of a series of overlapping sequences that comprise the entire desired sequence.
sequencing of overlapping, subcloned restriction fragments derived from the larger fragment.
Correct! It is important that a fully overlapping sequence with no gaps be established by this method.
sequencing of a set of terminal deletions of the large fragments to generate an overlapping set of nested sequences.
Correct! Exonuclease III and BAL31 are frequently used to generate the required set of deletion derivatives.

Types of polynucleotide that can be directly sequenced by the enzymatic approach include
RNA.
Correct! The reactions require the use of reverse transcriptase rather than DNA polymerase.
single-stranded DNA.
Correct! Vectors based on the single-stranded bacteriophage M13 were designed to facilitate this sequence analysis approach.
double-stranded DNA cloned in a vector.
Correct! The DNA must generally be denatured prior to the initiation of the sequence determination reaction.
PCR-amplified DNA.
Correct!

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.
False! Since the triplet code has been elucidated, it is easy to convert the nucleotide cDNA sequence to a predicted amino acid gene product sequence.
the site of transcription initiation.
Correct! Most routinely cloned cDNA molecules do not include the extreme 5' terminus of the mRNA molecule.
splicing and processing sites within the mRNA molecule.
Correct! Although a computer program might evaluate a sequence for the presence of sequences characteristic of processing junctions, these sites must be confirmed as valid by comparison with geneomic sequence or by mapping experiments.
regulatory sequences that influence mRNA expression levels.
Correct! The promoter and associated regulatory sequences are rarely included within the cloned cDNA molecule.