DNA sequencing

Question 1

describe dideoxy chain termination

Answer 1

aka Sanger sequencing
developed to sequence DNA in 1977
developed by Fred Sanger
very robust = low error rate, highly reliable

Question 2

sequencing by ABI 3730

Answer 2

sample is prepared by dideoxy chain termination on large scale by robotics

read length of up to 900bp- 99.95% accuracy

handles 48 or 96 samples simultaneously

over 1000 samples per day

only performs separation of LABELLED dna

Question 3

ABI 3730 automated DNA sequencing

Answer 3

used to sequence human genome
produced 23 thousand million bases of sequence
took 13 years
2.7 billion to complete

Question 4

how does sequencing by dideoxy chain termination work?

Answer 4

1. template
2. enzymatic sequencing section (DNA polymerase makes multiple copies of DNA)
3. size separation of products by capillary electrophoresis - sorting by size
4. detection of reaction products (sequential detection of terminating nucleotide to identify the base)
5. readout of sequence - reconstructing the sequence

Question 5

similarity of PCR and dideoxy chain termination

Answer 5

• some protocols cycle through repeated temperatures but only use a single forward primer

amplification is limited and not exponential

always uses a DNA polymerase

Question 6

sequencing by dideoxy chain termination - the reaction

Answer 6

sequencing reaction
strand seperation
annealing primer
extension 
chain termination

Question 7

describe sequencing using DNA polymerase

Answer 7

• DNA is mixed with reaction components including dideoxy and dideoxy-nucleotides

1. single strand oligonucleotide is bound to template
2. polymerase recognises the DNA structure, then forms initation complex

Question 8

describe requirements of DNA dependent DNA polymerase

Answer 8

1. template strand that extends beyond a primer
2. free 3’ OH group on primer
3. all 4 deoxy nucleotide triphosphate
4. Mg2+ ions

Question 9

describe final step: chain termination requirements

Answer 9

1. template strand extends a primer forming partial duplex
2. free 3’ OH group on primer
3. all 4 deoxy nucleotide triphosphates
4. all 4 dideoxy nucleotide triphosphates
5. mg2+ ions

Question 10

what is DNA elongation terminated by

Answer 10

addition of dideoxynucleotide as it has position 3 OH group missing which prevents extension

Question 11

describe sequencing using DNA polymerase

Answer 11

1. polymerase commences elongation from 3’ terminus
2. as enzyme encounters a particular nucleotide in sequence it picks out and incorporates complementary nucleotide into elongating strand

Question 12

how do reaction products vary in length terminated by ddNTP

Answer 12

• where ddCTP is incorporated = represents all positions in sequence where cytosine occurs
• if all 4 dideoxy nucleotides are present in the reaction the population of molecules produced represent all possible positions in sequence from same point to end

Question 13

describe size separation by gel electrophoresis

Answer 13

• nucleic acid passes through gel matrix by applying a voltage across two electrodes
• negatively charged nucleic acid migrates towards positive electrode
• matrix retards molecule according to size
• those larger are retarded to a greater extent and move through matrix more slowly

Question 14

how is the sequence determined

Answer 14

sequence is determined by direct comparison of lengths of products terminated by each of the 4 dideoxy nucleotides

Question 15

what does a measurement of fluorescence do

Answer 15

generates trace and base calling is automated

Question 16

how is DNA sequencing by dideoxy chain termination routinely used

Answer 16

health = test for specific genetic mutations in patients with suspected genetic diseases

• used to confirm all types of mutation
• identifying HIV haplotypes resistant to anti-retrovirals to determine therapy haart

Question 17

how is DNA sequencing by dideoxy chain termination still used

Answer 17

mammalian and pathogen gene sequencing
clone or PCR amplicon sequencing to confirm clones sequence or site-directed mutagenesis
walking a gene to identify a causative mutation in gene studies
confirmation of causative variants associated with genetic disease following association study