DNA Sequencing

Question 1

What is a type of DNA sequencing?

Answer 1

Dideoxy-chain termination

Question 2

What is another name for dideoxy-chain termination?

Answer 2

Sanger sequencing

Question 3

When was sanger sequencing developed?

Answer 3

In 1977, developed by Nobel Prize winner Fred Sanger

Question 4

Is Sanger sequencing still used?

Answer 4

The method is still used today. The technology has improved, the technique modified and semi-automated. It is a very robust technique with low error rate therefore reliable.

Question 5

How does automated DNA sequencing by ABI 3730?

Answer 5

• Samples prepared by dideoxy chain termination on a large scale by robotics
• Has read length of up to 900bp and 99.95% accuracy
• Handles 48 or 96 samples simultaneously
• It samples about 1000 samples per day
• Only performs the separation of labelled DNA and determines the sequence
• This is used to sequence the human genome producing 23 giga bases of sequence but took 13 years and $2.7 billion to complete

Question 6

Describe the process of sequencing by dideoxy chain termination

Answer 6

1. Template
2. Enzymatic Sequencing reaction
3. Size Separation of products by capillary electrophoresis
4. Detection of reaction products
5. Readout of sequence

Question 7

What happens during enzymatic sequencing reaction?

Answer 7

DNA polymerase makes multiple copies of the DNA

Question 8

How are products sorted by capillary electrophoresis?

Answer 8

Sorting by size

Question 9

How does detection of reaction products work?

Answer 9

Sequential Detection of the terminating nucleotide to identify the base

Question 10

What happens to the sequence at the end of dideoxy chain termination?

Answer 10

Re-constructing the sequence

Question 11

What enzyme does the traditional dideoxy nucleotide sequencing by strand termination method use?

Answer 11

It uses an enzyme called DNA dependent DNA polymerase to make copies of the complementary strand of a DNA template.

Question 12

How is the original sequence reconstructed from the readout?

Answer 12

Uses a separation step in which the molecules produced are sorted by size and since individual molecules are terminated by a particular dedoxynucleotide determined by the sequence, the original sequence can thus be reconstructed from the readout.

Question 13

What is similar between all dideoxy sequencing reaction?

Answer 13

Some protocols cycle through repeated temperatures but only uses a single forward primer - amplification is limited and not exponential. It also uses a DNA polymerase if the cycle is performed a thermostable polymerase so would be necessary and is usually used.

Question 14

How does DNA polymerase start?

Answer 14

1. A single stranded oligonucleotide (primer) is bound to the template
2. The polymerase recognises the DNA structure, then forms an initiation complex.
3. This commences elongation from the 3’ terminus of the primer in a 5’ to 3’ direction.

Question 15

What does DNA dependent DNA polymerase require during the elongation of sanger sequencing?

Answer 15

1. A template strand that extends beyond a primer
2. Free 3’ OH group on the primer
3. All 4 Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
4. Mg2+ ions

Question 16

What is required for chain termination in sanger sequencing?

Answer 16

1. A template strand that extends a primer forming a partial duplex
2. Free 3’OH group on the primer
3. All 4 deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
4. All 4 dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP)
5. Mg2+ ions

Question 17

Explain elongation in sanger sequencing

Answer 17

From the 5’ prime end, there is elongation in the 5’ to 3’ direction. At the 3’ OH end, a H+ and inorganic pyrophosphate

Question 18

How is DNA elongation terminated?

Answer 18

By the addition of dideoxynucleotide

Question 19

How do dideoxynucleotides prevent elongation?

Answer 19

The 3’OH group is missing which prevents the connection between H+ and pyrophosphate.

Question 20

Summarise the sequencing using DNA polymerase

Answer 20

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

Question 21

Why is it important to have all ddNTP?

Answer 21

• Products where a ddCTP is incorporated therefore represent all positions within the sequence where a “cytosine” occurs.
• Since all four labelled dideoxy nucleotides are present in the reaction the population of molecules produced represent all possible positions in the sequence from the same point to the end.

Question 22

What does ordering these molecules by size allow?

Answer 22

It allows us to determine the sequence of the new strand

Question 23

How does size separation happen? Explain how it happens

Answer 23

It can be done by gel electrophoresis.

• The nucleic acid passes through a gel matrix by applying a voltage across two electrodes.
• Negatively charged nucleic acid migrates towards the positive electrode.
• The matrix retards the molecules according to their size
• Those that are larger are retarded to a greater extent and as a consequence move through the matrix more slowly

Question 24

How is the sequence determined?

Answer 24

The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.

Question 25

What does the DNA dependent DNA polymerase generate?

Answer 25

Measurement of fluorescence generates a trace and base calling is automated

Question 26

When is DNA sequencing by dideoxy chain termination used?

Answer 26

- Health | - In reasearch

Question 27

When is Sanger sequencing used in health?

Answer 27

- Confirmatory test for specific genetic mutations in patients with suspected genetic diseases. - Identifying HIV haplotypes resistant to anti-retrovirals HAART

Question 28

What types of mutations does Sanger sequencing identify? And what is the one expection?

Answer 28

Silent, Missense, Nonsense, Truncating, Indel, and mis-splicing. The one exception is low frequency mosaicism

Question 29

When is Sanger Sequencing used in research?

Answer 29

- Mammalian and Pathogen Gene sequencing - 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