Molecular Diagnostics DNA Sequencing Flashcards
Terminology:
Method that allows us to determine the exact nucleotide sequence of a DNA fragment
DNA sequencing
TRUE or FALSE:
DNA sequencing applications include diagnostic, forensic, research, and biotechnology
TRUE
What are the 3 major methods of DNA sequencing?
-Direct sequencing (Sanger sequencing)
-Pyrosequencing
-Next-Generation sequencing (NGS)
DNA sequencing method:
-Most specific method for identifying genetic mutations (lesions) or polymorphisms
Direct sequencing
DNA sequencing method developed by Frederick Sanger
Sanger sequencing
Another name for Sanger sequencing
Dideoxy Chain Termination
Terminology:
Variances in DNA sequence that are found in 1-2% of a given population
Polymorphism
Essential components for Sanger sequencing (5)
- Template DNA
- DNA polymerase
- DNA primer
- dNTPs (DNA building blocks)
-ddNTPs (dideoxynucleotides)
Deoxyribose or Dideoxyribose?
Deoxyribose
Deoxyribose or Dideoxyribose?
Dideoxyribose
TRUE or FALSE:
Dideoxynucleotides are used to terminate DNA synthesis
TRUE
How many tubes are involved in the traditional process for Sanger sequencing?
4 tubes (one for each dNTP)
What are the first 7 in this DNA sequence?
T C C T A A G
How is the sequence ladder read? (top = closest to wells)
a. Top to bottom
b. Bottom to top
b. Bottom to top
What other Sanger sequencing methods are there?
- Single tube in which all components are added to one tube and electrophoresed in one lane
- Automated method using capillary electrophoresis
DNA sequencing method used to determine DNA sequence without making a sequencing ladder. Does not use gels, fluorescent dyes, or ddNTPs.
Pyrosequencing
What does pyrosequencing use?
Light generated when nucleotides are added to a growing strand of DNA
Mutation detection and infectious disease typing are applications of which type of DNA sequencing?
Pyrosequencing
The following are essential components for which method of DNA sequencing:
- DNA template
- Sequencing primer
- DNA polymerase
- Enzymes (sulfurylase, luciferase)
- Substrates (APS, luciferin)
Pyrosequencing
What is the pyrosequencing graph called?
Pyrogram
DNA sequencing method that sequences large numbers of templates simultaneously
Next-Generation sequencing
Ion-conductive sequencing and reversible dye terminator sequencing are clinical technologies of which DNA sequencing method?
Next-Generation sequencing
The following are the basic principles of which DNA sequencing method:
- DNA is fragmented into multiple pieces and adapters are added to the ends of the fragments
- This collection of DNA fragments to be sequenced is called a sequencing library
- The library is sequenced using a concept similar to capillary sequencing
- Then the whole sequence is used for data analysis
Next-Generation analysis
