Molecular Diagnostics Flashcards
Terminology:
Method that allow us to determine the exact nucleotide sequence of a DNA fragment
DNA sequencing
The DNA sequencing method developed by Frederick Sanger that is the most specific method for identifying genetic mutations or polymorphisms
Direct Sequencing - Sanger Sequencing
Other names for Direct Sequencing (2)
- Sanger Sequencing
- Dideoxy Chain Termination Sequencing
Terminology:
Variances in DNA sequence that are found in 1-2% of a given population
Polymorphism
This DNA sequencing method is especially useful when looking for changes affecting only one or two nucleotides
Direct sequencing
Essential components for Sanger sequencing (5)
- Template DNA
- DNA polymerase
- DNA primer
- dNTPs (DNA building blocks)
- ddNTPs (dideoxynucleotides)
Dideoxyribose or Deoxyribose?
Deoxyribose
Dideoxyribose or Deoxyribose?
Dideoxyribose
TRUE or FALSE:
Deoxynucleotides are used to terminate DNA synthesis in Sanger sequencing
FALSE - Dideoxynucleotides
In what direction is the sequence ladder read? (top is closest to wells)
a. Bottom to top
b. Top to bottom
a. Bottom to top
What would be the first 7 nucleotides in this sequence?
T C C T A A G
DNA sequencing method that determines sequence without making a sequencing ladder.
-No gels, ddNTPs, or fluorescent dyes
Pyrosequencing
What is measured in pyrosequencing and how?
Light is measure when generated as nucleotides are added to the DNA strand.
The following are applications for which DNA sequencing method:
- Mutation detection
- Infectious disease typing
Pyrosequencing
The following are essential components for which DNA sequencing method:
- DNA template
- Sequencing primer
- DNA polymerase
- Enzymes (sulfurylase, luciferase)
- Substrates (APS, luciferin)
Pyrosequencing
The following process describes which DNA sequencing method:
- One dNTP is added to the reaction at a time
- If it binds to the strand, a luminescent signal is generated
- Generation of light = dNTP was added
- Light generation is recorded
Pyrosequencing
What is the pyrosequencing graph known as?
Pyrogram
DNA sequencing method that can take place in 4 separate tubes, a single tube, or using capillary electrophoresis
Sanger sequencing (Direct sequencing)
DNA sequencing method that sequences large numbers of templates carrying millions of bases simultaneously
Next Generation Sequencing (NGS)
Another name for Next generation sequencing
Massive parallel sequencing
The following describe 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 sequencing
