MLAB 2378 Fundamentals of Molecular Diagnostics Links

MLAB 2378 Fundamentals of Molecular Diagnostics

Unit 12 Objectives - DNA Sequencing

List 4 purposes that DNA sequencing information is routinely used for in the clinical laboratory.
List the two methods extensively utilized to sequence DNA.
Briefly describe the steps in the Maxam-Gilbert sequencing method.
Briefly describe the steps in the Sanger sequencing methods.
List the three denaturing conditions which prevent single strands of DNA from bonding with each other or folding up as they migrate through the gel.
List the three components of the stop buffer and the purpose of each.
State the importance of maintaining equal reaction time after the addition of polymerase in the Sanger method.
Given a sequencing ladder interpret the order of the nucleotides.
State the benfits of using heat stable recombinant polymerase enzymes and heat stable enzymes in the sequencing reaction.
State the advantages of using an automated fluorescent sequencing analyzer.
Describe how the use of four fluorescent dyes has enhanced the process of DNA sequencing.
Compare and contrast the dye primer and dye terminator methods of DNA sequencing.
List three methods which may be used to remove excess dye terminators from the reaction tube.
State the affect that formation of a secondary structure will have on the speed and quality of the sequence.
Briefly describe how fluorescently labeled nucleotides are detected in fluorescent detection equipment.
List three items that will impact the quality of an electropherogram.
Briefly describe the pyrosequencing reaction and state what it is most commonly used for.
Briefly describe the Bisulfate DNA sequencing method and state the primary use of this technique.
List the two groups who were in competition to map the human genome and the method used by each.
State the year that the first rough draft of the human genome was completed.
State the goal of the Human Haplotype Mapping project.
State the benefits that the Human Genome Project has provided the clinical laboratory.
Define or describe the terms listed in the "Unit 12 Vocabulary".

Sequencing Procedure

Double-stranded DNA is dissociated and a radioactive or fluorescent primer is annealed to the DNA (Figure 8-A); four separate reactions are performed to synthesize new DNA, each reaction contains all four deoxynucleotides and a small portion of one of the dideoxynucleotide bases (8-B); DNA is synthesized, terminating each time a ddNTP is incorporated (8-C); DNA from all four reactions is separated on a gel in side-by-side lanes to produce a sequence ladder (8-D); the sequence is read from the bottom up, and is the compliment (opposite) of the base identified in the gel. (Reference NBII)

Unit 12 Objectives - DNA Sequencing

Sequencing Procedure

Sequence ladder by radioactive sequencing compared to fluorescent peaks