Molecular Diagnostics DNA Sequencing

Question 1

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

Answer 1

DNA sequencing

Question 2

TRUE or FALSE:
DNA sequencing applications include diagnostic, forensic, research, and biotechnology

Answer 2

TRUE

Question 3

What are the 3 major methods of DNA sequencing?

Answer 3

-Direct sequencing (Sanger sequencing)
-Pyrosequencing
-Next-Generation sequencing (NGS)

Question 4

DNA sequencing method:
-Most specific method for identifying genetic mutations (lesions) or polymorphisms

Answer 4

Direct sequencing

Question 5

DNA sequencing method developed by Frederick Sanger

Answer 5

Sanger sequencing

Question 6

Another name for Sanger sequencing

Answer 6

Dideoxy Chain Termination

Question 7

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

Answer 7

Polymorphism

Question 8

Essential components for Sanger sequencing (5)

Answer 8

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

Question 9

Deoxyribose or Dideoxyribose?

Answer 9

Deoxyribose

Question 10

Deoxyribose or Dideoxyribose?

Answer 10

Dideoxyribose

Question 11

TRUE or FALSE:
Dideoxynucleotides are used to terminate DNA synthesis

Answer 11

TRUE

Question 12

How many tubes are involved in the traditional process for Sanger sequencing?

Answer 12

4 tubes (one for each dNTP)

Question 13

What are the first 7 in this DNA sequence?

Answer 13

T C C T A A G

Question 14

How is the sequence ladder read? (top = closest to wells)
a. Top to bottom
b. Bottom to top

Answer 14

b. Bottom to top

Question 15

What other Sanger sequencing methods are there?

Answer 15

• Single tube in which all components are added to one tube and electrophoresed in one lane
• Automated method using capillary electrophoresis

Question 16

DNA sequencing method used to determine DNA sequence without making a sequencing ladder. Does not use gels, fluorescent dyes, or ddNTPs.

Answer 16

Pyrosequencing

Question 17

What does pyrosequencing use?

Answer 17

Light generated when nucleotides are added to a growing strand of DNA

Question 18

Mutation detection and infectious disease typing are applications of which type of DNA sequencing?

Answer 18

Pyrosequencing

Question 19

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

Answer 19

Pyrosequencing

Question 20

What is the pyrosequencing graph called?

Answer 20

Pyrogram

Question 21

DNA sequencing method that sequences large numbers of templates simultaneously

Answer 21

Next-Generation sequencing

Question 22

Ion-conductive sequencing and reversible dye terminator sequencing are clinical technologies of which DNA sequencing method?

Answer 22

Next-Generation sequencing

Question 23

The following are the basic principles of 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 23

Next-Generation analysis