8P: Molecular genetics II

Question 1

Detection of unknown mutation

Answer 1

Whole-exome sequencing (WES)

Question 2

What is sequencing?

Answer 2

Determination of the nucleotide sequence of DNA fragments

Question 3

Sequencing methods

Answer 3

Maxam-Gilber and Sanger methods

Question 4

Sanger method

Answer 4

• Detection of point mutations
  1) PCR reaction with dNTP and ddNTP (dideoxyribonucleotides)
  2) Because of the absence of 3`OH, the synthesis ends when ddNTP is incorporated
  3) Shorter and longer fragments arise
  4) Capillary electrophoresis: All four ddNTPs fluorescently labeled (four colors)
  5) Laser reading: colored ladder peaks -> base sequence

Question 5

Aim of genotyping by sequencing

Answer 5

Not looking for a specific known mutation or polymorphism, but for sequence differences in a not too large, known gene

Question 6

Next generation sequencing (NGS)

Answer 6

A lot more nucleotides can be sequenced than when using traditional sequencing.
Used for research, whole genome sequencing

Question 7

Steps of NGS

Answer 7

1) Sequencing a primer hybridised to the template
2) Incorporation of each dNTP is accompanied by the release of pyrophosphate (PPi) group
3) PPi generated (multistep process) visible light can be detected and presented in the program
4) An enzyme degrades unincorporated dNTPs. Then next dNTP is added
5) Addition of dNTPs is performed one at a time, and the program generated from the visible light show the sequence

Question 8

What is RT-PCR used for?

Answer 8

Reverse transcriptase polymerase chain reaction
- For the analysis of mRNA expression
(But since mRNA is not stable enough, we need cDNA that can be analysed using a traditional PCR reaction)

Question 9

Advantage of real-time/quantitative PCR (qPCR)?

Answer 9

• Accurate quantification of the amplified DNA by means of fluorescence measurements
• Enables both quantitative analysis of DNA as well as determination of the amount of RNA

Question 10

Why is DNA fingerprinting used?

Answer 10

1) To determine who is the child´s father
2) Link suspects to a crime scene
3) Determine if a family relationship exist between two people