DNA SEQUENCING Flashcards
(22 cards)
What is DNA sequencing?
The process of determining the sequence of bases in a DNA molecule
DNA is encoded in the form of four bases: A, G, C, and T.
What are the four bases of DNA?
- Adenine (A)
- Guanine (G)
- Cytosine (C)
- Thymine (T)
What is the purpose of analyzing DNA fragments?
To determine the nucleotide sequence and the distribution and location of restriction sites.
Name one method of DNA sequencing.
- Chemical Cleavage Method
- Chain Termination Method
- Next Generation Sequencing: Pyrosequencing
What is the Maxam-Gilbert method?
A DNA sequencing method based on chemical modification of DNA and cleavage at specific bases.
What is required for the Maxam-Gilbert method?
- Radioactive labeling at one end
- Purification of the DNA fragment
What does the chemical treatment in the Maxam-Gilbert method generate?
Breaks at a small proportion of one or two of the four nucleotide bases.
What is a key feature of the Maxam-Gilbert method?
Base-specific cleavage of DNA by certain chemicals.
What is one advantage of the Maxam-Gilbert method?
Purified DNA can be read directly.
What is a disadvantage of the Maxam-Gilbert method?
It requires extensive use of hazardous chemicals.
What is the Sanger method also known as?
Chain Termination Method or Sanger Dideoxy Method.
What is the key principle of the Sanger method?
The use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.
What are the components required for the Sanger method?
- Single-stranded DNA template
- DNA primer
- DNA polymerase
- Radioactively or fluorescently labelled nucleotides
- Modified nucleotides that terminate DNA strand elongation
What happens to the DNA sample in the Sanger method?
It is divided into four separate sequencing reactions.
What does a dark band in a lane indicate in the Sanger method?
A DNA fragment resulting from chain termination after incorporation of a dideoxynucleotide.
What is a key feature of the Sanger method?
Uses dideoxy nucleotides to terminate DNA synthesis.
What is one advantage of the Sanger method?
Chain termination methods have greatly simplified DNA sequencing.
What is Pyrosequencing?
A DNA sequencing technology based on the sequencing-by-synthesis principle.
What is a key feature of Pyrosequencing?
Bases are added one at a time, and chemiluminescence is used to determine which base was added.
What is the benefit of Pyrosequencing compared to other methods?
It does not require electrophoresis or any other fragment separation.
What is solid phase sequencing?
A method where the template and primer are immobilized on solid support.
In solid phase sequencing, how is incorporation detected?
By addition of ATP sulfurylase and luciferase.
