21.2 DNA sequencing

Question 1

what is DNA sequencing?

Answer 1

process of determining the precise order of nucleotides within a DNA molecule

Question 2

what were initial methods of DNA sequencing?

Answer 2

bases were radioactively labelled and the gel electropheresis is carried out

Question 3

what replaced radioactive labels in modern DNA sequencing?

Answer 3

fluorescent tags

Question 4

what was the HGP?

Answer 4

the human genome project

massive intl project which mapped the entire human genome

Question 5

what was the initial timeline for the completion of HGP?

Answer 5

15 years

Question 6

why was the HGP achieved earlier than expected?

Answer 6

the automation of sequencing techniques
development of faster computers

Question 7

when was the first complete human genome sequence published?

Answer 7

2003

Question 8

what is the method by which DNA sequencing occurs by?

Answer 8

chain termination method

Question 9

step 1 of DNA sequencing

Answer 9

the DNA is mixed with:
- primer
- DNA polymerase
- excess free nucleotides
- terminator bases

Question 10

step 2 of DNA sequencing

Answer 10

mixture placed in a thermal cycler (PCR)

which causes the DNA to separate into single strands

eventually the primers bind to the strands

Question 11

step 3 of DNA sequencing

Answer 11

DNA polymerase builds up new DNA strands by adding nucleotides with complimentary bases to the single-stranded DNA

Question 12

what are attached to the terminators?

Answer 12

fluorescent tags

Question 13

step 4 of DNA sequencing

Answer 13

when a terminator base binds to it’s complimentary base, the sequence is ended

this leads to many fragments being formed of different lengths

Question 14

step 5 of DNA sequencing

Answer 14

the DNA fragments are then separated by gel electrophoresis

the fluorescent markers on the terminator bases are used to identify the final base of each fragment

Question 15

how can the base sequence be identified from gel electrophoresis?

Answer 15

the first base (the smallest fragment) will have it’s band at the top of the plate (positive end)

by reading the preceding bands, you can identify the base sequence

Question 16

how are the fluorescence of the terminators detected?

Answer 16

using lasers

Question 17

what is the product of the chain termination method?

Answer 17

the order of bases shown in capillary sequencing shows the sequence of the complimentary strand of DNA which is then used to form the original DNA strand

Question 18

how is a genome resembled?

Answer 18

they are compared with all the fragments in the database and areas of overlap are found

Question 19

overview of DNA sequencing

Answer 19

Question 20

what has replaced the use of gel plates?

Answer 20

a plastic side called a flow cell

Question 21

what is massively parallel sequencing?

Answer 21

fragments of DNA are attached to a flow cell and replicated using PCR to form clusters of DNA fragments

then the coloured terminators are added
at the same time images are taken

Question 22

adv of new DNA sequencing

Answer 22

extremely efficient
very fast

Question 23

what ways can DNA sequencing be faster?

Answer 23

high throughput sequencing
(pyrosequencing)

entire genome sequencing

Question 24

how is entire genome sequencing carried out?

Answer 24

genome is cut into smaller pieces using restriction endonucleases

fragments are inserted into BACs which are man made plasmids

BACs are inserted into bacteria

the bacteria divide to create colonies of cloned cells

DNA is extracted from each colony and cut up using restriction endonucleases

DNA fragments are sequenced

then DNA fragments are put together using overlapping regions to complete the entire genome

Question 25

how is DNA sequencing become automised?

Answer 25

Question 26

what is high-throughput sequencing?

Answer 26

faster sequencing method which is also cheaper

Question 27

outline pyrosequecning

Answer 27

section of DNA separated into two strands

each band binds to small bead

PCR amplifies the DNA fragments on the beads

each bead is put into a well

free nucleotides are added to wells containing the beads

the wells have enzymes which emit light when when a free nucleotide binds to DNA strand

if the nucleotide is repeated, the light emitted is greater

computer analyses data to find base sequence