DNA sequencing and genomics

Question 1

DNA sequencing is used for?

Answer 1

determining gene sequences that provide basis for investigating the genetics of health and disease
knowing gene sequence is critical for other experiments (such as constructing primers for PCR reactions)

Question 2

the purpose of DNA sequencing is?

Answer 2

is used to determine the sequence of nucleotides in a stand of DNA

Question 3

why are modified nucleotides, dideoxynucleotides (ddNTPs) used?

Answer 3

they are missing the OH group on the 3’ carbon thus are unable to created a new 5’ to 3’ phosphodiester bond which allows people to control the termination of replication

Question 4

in step one of DNA sequencing, what happens?

Answer 4

the DNA strand of interest is denatured using NaOH solution to create a ssDNA strand to be used for replication

Question 5

in step two of DNA sequencing, what happens?

Answer 5

the ssDNA strand is added to a solution containing:
-radiolabeled DNA primer that is complementary ot the gene of interest
-DNA polymerase
-all four dNTPs (dATP, dTTP, dCTP, dGTP)
-small quantity of single ddNTP
this step yields a population of newly synthesized DNA fragments that are varying sizes and complimentary to the template strand and covalently bonded to a radio-labeled primers at the 5’ end

Question 6

why are the newly synthesized DNA fragments different sizes?

Answer 6

the insertion of ddATP is random in the growing chain

Question 7

the third step of DNA sequencing, what happens?

Answer 7

three more separate solution reactions (same as step 2) are done for the remaining nucleotides (ddCTO, ddGTP, ddTTP)
*ddATP was done in step 2

Question 8

in the fourth step of DNA sequencing, what happens?

Answer 8

each solution containing specific dNTP and ddNTP are placed in their own well and ran under gel electrophoresis
*each reaction are run in their own lane

Question 9

in step five of DNA sequencing, what happens?

Answer 9

the gel is transferred to a polymer sheet and autoradiography is used to identify the strands in the gel

Question 10

the smallest fragments will migrate furtherest on the well and only have what attached to it?

Answer 10

a single ddNTP attached to it

Question 11

the second smallest fragment has what attached to it?

Answer 11

it has a primer with two bases attached

Question 12

reading the well from bottom to top indicates the sequence of the complimentary strand in what direction?

Answer 12

5’ to 3’ direction

Question 13

the first way genomic sequencing is done is by?

Answer 13

• generating a genetic linkage map with several hundred markers per chromosome
• the map is refined to a physical map by preparing YAC and BAC libraries containing large chromosomal fragments
• the library is put in order then gradually cloned into libraries containing smaller fragments
• each of these fragments are eventually sequence and assembled into an overall sequence

Question 14

the second way genomic sequencing is done by?

Answer 14

• whole-genome shotgun approach where the chromosomes are cut into small fragments which are cloned and sequenced
• the strategy skips generating maps and requires more extensive analysis of sequencing data by computers in order to align fragments

Question 15

genome sequencing is used to make predictions on?

Answer 15

how many genes there are in a certain organisms, where they are located, how expression is controlled, and how genome is organized

Question 16

genome sequencing is also used for?

Answer 16

studying genetic variation within and across species