DNA sequencing

Question 1

define DNA sequencing

Answer 1

technique used to determine the exact sequence of bases in a DNA molecule

Question 2

define terminator base

Answer 2

modified versions of the four nucleotide bases which stop DNA synthesis when they are read

Question 3

define high-throughput sequencing

Answer 3

sequencing DNA and RNA in a more rapid and cost-effective manner

Question 4

explain the first step of DNA sequencing (mixing)

Answer 4

DNA to be sequenced mixed in thermocycler with:
- DNA primers
- DNA polymerase
- terminator bases
- excess of nucleotides

Question 5

explain the second stage of DNA sequencing (first heating)

Answer 5

thermocycler heated to 95ºC which denatures the DNA - splits into 2 strands

Question 6

explain the third stage of DNA sequencing (first heat reduction)

Answer 6

thermocycler temp reduced to 50ºC so primer can anneal to the DNA

Question 7

explain the fourth stage of DNA sequencing (second temperature increase)

Answer 7

thermocycler temp reduced to 60ºC
- DNA polymerase joins free nucleotides to exposed bases (CBP)
- when terminator base is added, synthesis is terminated

Question 8

what features of a terminator base make it so no other nucleotide can bind to it?

Answer 8

• carbon 3 on ribose ring has H instead of OH

Question 9

why are the DNA fragments in DNA sequencing different lengths?

Answer 9

• terminator bases added at random
• there are lower amounts of terminator bases

Question 10

explain the fifth stage of DNA sequencing (GEP)

Answer 10

DNA fragments are separated by length through gel electrophoresis
- fluorescent markers are used to identify final base on each fragment

Question 11

what does the order of bases show after gel electrophoresis?

Answer 11

the sequence of new, complementary DNA
- used to build up sequence of original DNA strand

Question 12

describe 3 reasons for developing new DNA sequencing technologies.

Answer 12

• allows more efficient sequencing, with lower cost
• able to carry out sequencing in 24h, used to be 13 year long process
• we use information from genomes & carry out large scale genome studies

Question 13

explain why the correct sequence of amino acids is not always provided in a protein

Answer 13

some genes can code for many different proteins

Question 14

define DNA primer

Answer 14

short nucleic acid sequence that provides a starting point for DNA synthesis