Sanger DNA Sequencing Flashcards
Who is Sanger
Worker out the structure of insulin
Determing sequences of DNA
What are the building blocks of DNA?
Adnine
Thymine
Cytosine
Guanine
Phosphate backbone
What is DNA polymerase?
The primary enzyme which catalyses the linkage of the 3’ hydroxyl group of the end nucleotide to the 5’ phosphate of nucleotide to be added
What do dideoxyribonucleotide triphosphates lack?
Oxygen on the 3’
What are dideoxy building blocks?
ddATP
ddCTP
ddGTP
ddTTP
What are ddATP, ddCTP, ddGTP and ddTTP building blocks for?
Dideoxynibonucleotides
What is the first stage of Sanger sequencing?
Denature dsDNA using heat
What is the second stage of Sanger sequencing?
Make multiple copies of a segment
What is the third stage of Sanger sequencing?
Attach a primer
What is the fourth stage of Snager sequencing?
Add four polymerase solutions
What is the fifth sage of Sanger sequencing?
Grow complementary chains until termination dye
What is the sixth stage of Snager sequencing?
Denature the grown chains?
What is the seventh stage of the Snager sequence?
Electrophorese the four solutions
Which way do you read the DNA strand sequence - 5’ to 3’ or 3’ to 5’?
5’ to 3’
What are the three updates of Snager sequencing?
- PCR with fluorescent chain-terminating ddNTP’s
- Size seperation by capillary gel electrophoresis
- Laser excitation & detection by sequencing machine
