DNA Sequencing

Question 1

DNA Sequencing

Answer 1

Process involved in determining exact sequence of nucleotides in piece of DNA –> used by Human Genome Project; dideoxy termination sequencing method developed in 1975 by Frederick Sanger

Question 2

Dideoxynucleotide Triphosphate

Answer 2

Regular deoxy nucleotide except missing oxygen at 3’ carbon (no OH group) –> cannot add another nucleotide due to lack of OH at 3’ - stops replication

Question 3

Sanger Sequencing Components

Answer 3

Single stranded DNA template (uses heat to separate), radioactive primers, DNA polymerase, dideoxynucleotide triphosphates (ddA, ddT, ddG, ddC), regular deoxynucleotides

Question 4

Process

Answer 4

1. ddA, ddT, ddG, ddC added individually into 4 separate tubes with all other components
2. DNA replication occurs in each tube
3. When dideoxynucleotide incorporated, replication STOPS, creating DNA fragments of different of many lengths in each test tube
4. Gel electrophoresis used to separate all fragments - each tube placed into own lane

Question 5

Modern Sequencing

Answer 5

Exactly same as Sanger method except:
- 4 different coloured fluorescent dyes used to label 4 different dideoxynucleotides in single tube
- 4 fluorescent dideoxynucleotides run on one lane in gel electrophoresis
- Fragments still separated by size but now coloured

Question 6

Reading Gel

Answer 6

Read from shortest to longest (positive end to negative end) to map in 5’–>3’ direction