DNA Sequencing Flashcards
What bonds join nucleotides together?
Covalent Phosphodiester bonds
How are nucleotides joined together?
5’ - phosphate group of one nucleotide joined to 3’ - hydroxyl group of the next nucleotide
What are the preliminary steps of Conventional Sequencing of DNA?
- Isolation of dsDNA from tissue
- Preparation of dsDNA for sequencing
What are the FOUR steps in the Main Procedure for the Conventional Sequencing of DNA?
- Sequencing - chain fragmentation or elongation
- Separation of the generated fragments
- Detection: radiography or fluorescence
- Reading and Alignment of target sequence
What chemical breaks the nucleotide chain at Guanine?
DMS - dimethyl sulfate
What chemicals break the nucleotide chain at Guanine AND Adenine?
DMS + formic acid
What is the base catalyst involved in chemical chain breakage of nucleotides?
Piperidine
What chemical breaks the nucleotide chain at Cysteine AND Thymine?
Hydrazine
What chemicals break the nucleotide chain at Cysteine?
Hydrazine + salt
What radioactive label is introduced at Primer’s 5’-end in Sanger Method?
32P
What colour is the ddCTP fluorescent dye involved in Sanger Method?
Yellow
What colour is the ddGTP fluorescent dye involved in Sanger Method?
Green
How many bases can Automated Sanger Methods sequence?
500, 000 per day
What are the comparisons between the Maxam-Gilbert and Sanger methods?
What is the benefit of NGS?
Like running a huge number of Sanger reactions in parallel
