Molecular Diagnostics

Question 1

Terminology:
Method that allow us to determine the exact nucleotide sequence of a DNA fragment

Answer 1

DNA sequencing

Question 2

The DNA sequencing method developed by Frederick Sanger that is the most specific method for identifying genetic mutations or polymorphisms

Answer 2

Direct Sequencing - Sanger Sequencing

Question 3

Other names for Direct Sequencing (2)

Answer 3

• Sanger Sequencing
• Dideoxy Chain Termination Sequencing

Question 4

Terminology:
Variances in DNA sequence that are found in 1-2% of a given population

Answer 4

Polymorphism

Question 5

This DNA sequencing method is especially useful when looking for changes affecting only one or two nucleotides

Answer 5

Direct sequencing

Question 6

Essential components for Sanger sequencing (5)

Answer 6

• Template DNA
• DNA polymerase
• DNA primer
• dNTPs (DNA building blocks)
• ddNTPs (dideoxynucleotides)

Question 7

Dideoxyribose or Deoxyribose?

Answer 7

Deoxyribose

Question 8

Dideoxyribose or Deoxyribose?

Answer 8

Dideoxyribose

Question 9

TRUE or FALSE:
Deoxynucleotides are used to terminate DNA synthesis in Sanger sequencing

Answer 9

FALSE - Dideoxynucleotides

Question 10

In what direction is the sequence ladder read? (top is closest to wells)
a. Bottom to top
b. Top to bottom

Answer 10

a. Bottom to top

Question 11

What would be the first 7 nucleotides in this sequence?

Answer 11

T C C T A A G

Question 12

DNA sequencing method that determines sequence without making a sequencing ladder.
-No gels, ddNTPs, or fluorescent dyes

Answer 12

Pyrosequencing

Question 13

What is measured in pyrosequencing and how?

Answer 13

Light is measure when generated as nucleotides are added to the DNA strand.

Question 14

The following are applications for which DNA sequencing method:
- Mutation detection
- Infectious disease typing

Answer 14

Pyrosequencing

Question 15

The following are essential components for which DNA sequencing method:
- DNA template
- Sequencing primer
- DNA polymerase
- Enzymes (sulfurylase, luciferase)
- Substrates (APS, luciferin)

Answer 15

Pyrosequencing

Question 16

The following process describes which DNA sequencing method:
- One dNTP is added to the reaction at a time
- If it binds to the strand, a luminescent signal is generated
- Generation of light = dNTP was added
- Light generation is recorded

Answer 16

Pyrosequencing

Question 17

What is the pyrosequencing graph known as?

Answer 17

Pyrogram

Question 18

DNA sequencing method that can take place in 4 separate tubes, a single tube, or using capillary electrophoresis

Answer 18

Sanger sequencing (Direct sequencing)

Question 19

DNA sequencing method that sequences large numbers of templates carrying millions of bases simultaneously

Answer 19

Next Generation Sequencing (NGS)

Question 20

Another name for Next generation sequencing

Answer 20

Massive parallel sequencing

Question 21

The following describe which DNA sequencing method:
- DNA is fragmented into multiple pieces and adapters are added to the ends of the fragments
- This collection of DNA fragments to be sequenced is called a sequencing library
- The library is sequenced using a concept similar to capillary sequencing
- Then the whole sequence is used for data analysis

Answer 21

Next generation sequencing