DNA sequencing/ Sanger Sequencing/ Dideoxychain Termination

Question 1

Describe how DNA sequencing has become automated?

Answer 1

• Machine most commonly used is ABI 3730
• Samples prepared by dideoxy chain termination on a large scale by robotics
• Read length up to 900 base pairs + 99.95% accuracy
• Only performs the seperation of labelled DNA and determines the sequence - requires considerable hands-on manipulation

Question 2

What are the steps involved in dideoxy chain termination?

Answer 2

1. Generating template (this can either be a clone (plasmid) or an amplicon from PCR
2. Sequencing reaction: DNA Polymerase makes copies of the template
3. Seperation of the DNA strands by size carried out by capillary electrophoresis
4. Detection of reaction particles - sequential detection of terminating nucleotide to identify the base
5. Readout of sequence - where the sequence is reconstructed

Question 3

How does PCR and dideoxy chain termination differ?

Answer 3

• Similar cycle thrpugh repeated temperatures, however dideoxy chain chain termination only uses a single forward primer - this means the amplification is LINEAR and not exponential
• Both use DNA polymerase (thermostable)

Question 4

What are the steps involved in sequencing by dideoxy chain termination?

Answer 4

1. Strand seperation
2. Anenaling primer
3. Extension
4. Chain Termination

Question 5

Explain the first two steps of dideoxy chain termination

Answer 5

First two steps are:

1. Strand seperation
2. Annealing of primers

• DNA sample is denatured to seperate strand and mixed with dideoxy nucleotides
• Single stranded oligonucleotide primers which are complementary to the sequence are hybridised and act as a template for DNA polymerase
• DNA polymerase recognises the partial ds molecule with 3’ OH group = forms initiation complex

Question 6

Explain the third step of the sequencing reaction

Answer 6

Third step is:

• Extension

Occurs by the action of DNA polymerase which requires:

1. Template strand that extends beyond a primer
2. Free 3’ OH group on the primer
3. All 4 deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
4. Buffer to stabillise the pH
5. Mg2+

Question 7

Explain the fourth step of the sequencing reaction?

Answer 7

The fourth step is:

• Elongation
  
  • DNA elongation is terminated by addition of a dideoxynucleotide (ddATP, ddGTP, ddCTP, ddTTP) these are at a low molar excess compared to deoxynucleotide triphosphates.
  • This can no longer cause progression in the reaction due to the missing -OH group on the 3’/carbon 3
  • This results in dissociation of DNA polymerase from the strand

Question 8

What occurs as a consequence of adding dideoxy molecules into the sequencing reaction?

Answer 8

• e.g products where a ddCTP is incorporated represent all positions in the sequence where a ‘cytosine’ occurs
• Since all four fluorescently labelled dideoxynucleotides are present in the reaction, the population of molecules produces represent all possible reactions in the sequence from the same point to the end
• Ordering these molecules by size allows us to determine the sequence of the new strand

Question 9

Explain the stages after addition of the dideoxy nucleotide triphosphates

Answer 9

SIZE SEPERATION is carred out

• Size seperation is carried out via gel capillary electrophoresis
• NA passes through gel matrix applying voltage across 2 electrodes
• Negatively charges NAs migrate towards negative electrode
• The matrix will retard molecules according to size
• Larger molecules are retarded to a greater extent and move more slowly
• Smallest molecule will be closest to primer and 5’ end, larger molecules will be slower and closer to 3’ end

Question 10

How is the sequence determines after size seperation?

Answer 10

Sequence is determines by direct comparison of lengths of products terminated by each of the four dideoxynucleotides

Question 11

How is DNA sequencing/ dideoxy chain termination used in health?

Answer 11

• Confirmatory test for genetic mutations in patients with suspected diseases
  
  • Used to confirm all types of mutations e.g
    
    • Silent, missense, nonsense, truncatinf, indel + mis-splicing
    • HOWEVER = not low frequency mosacism (not used in DNA seq as below sensitivity to detect this as mosaicisms is where some cells have the mutation and others dont)
  • Identifying HIV haplotypes resistant to anti-retroviral HAART

Question 12

How is dideoxy chain termination used in research?

Answer 12

• Mammalian and pathogen gene seq
• Clone or PCR Amplicon sequencing to confirm a clones sequence or site-directed mutagenesis
• “Walking” a gene to identify a causative mutation in candidate gene studies
• Confirmation of causative variants associated with genetic disease following association study