DNA sequencing

Question 1

what is DNA sequencing?

Answer 1

= determining the order of nucleotides in a sample of DNA

- mapping out sections of DNA that can be compared to identify ‘dodgy’ alleles

Question 2

what are the uses for DNA sequencing?

Answer 2

• identifying mutations
• compare DNA from different organisms
• identify genetic disease
• maternity and paternity tests
• compare species to track evolutions

Question 3

what are the 4 key ingredients in DNA sequencing?

Answer 3

DNA sample (template)
- piece of DNA containing gene/section to be sequenced

primer

• copies of snippets of DNA that act as markers to show where we want to sequence the DNA
• complimentary to target sequence

nucleotides

• normal free nucleotides
• altered nucleotides = OH replaced for O; fluro dye added

DNA/taq polymerase
- enzyme to catalyze reaction

Question 4

what is the first step of DNA sequencing?

Answer 4

• DNA primer, free nucleotides + one type of modified nucleotide placed in ‘tube”

Question 5

what is the second step of DNA sequencing?

Answer 5

heat = 96

- DNA is denatured and separates

Question 6

what is the third step of DNA sequencing?

Answer 6

cool = 50

• primer attaches to sections of DNA
• tells Taq polymerase where to start

Question 7

what is the fourth step of DNA sequencing?

Answer 7

warm = 60
- Taq polymerase builds copies of DNA, stopping at each occurrence of modified nucleotide
DNA is copied into smaller strands

Question 8

what is the fifth step of DNA sequencing?

Answer 8

repeat with each modified nucleotide

Question 9

what is the sixth step of DNA sequencing?

Answer 9

shortened sections of DNA move past a laser and each fluro marker is identified

Question 10

what is the seventh step of DNA sequencing?

Answer 10

machine interprets data into a sequence

Question 11

describe sangers method

Answer 11

• synthetic nucleotides that lack OH group are added to the growing strand = deoxyribonucleotides
• synthetic nucleotides stop the elongation of the sequence because there is no OH group for the next nucleotide to attach to = each nucleotide of DNA = different lengths of DNA
• separated through gel electrophoresis
• knowing which base was added = determine nucleotide order

Question 12

what is the role of chain-terminating nucleotides?

Answer 12

• halts the ability of the polymerase to attach free nucleotides
• means polymerase can no longer synthesize bases
• produces lots of different strands of different lengths

Question 13

how can we determine which nucleotide is which in sangar sequencing?

Answer 13

• attached to chain-terminating nucleotides there is a fluorescent dye
• when passed through a detector reflects back a color so the computer can detect and associate that color with each base