detecting variants in DNA: DNA sequencing Flashcards
(25 cards)
What is the purpose of DNA variant analysis?
Clinical genetics, pharmacogenetics, genetic mapping, genome wide association studies (GWAS), identification
These applications help in understanding genetic diseases and tailoring medical treatments.
What is the difference between mutation and polymorphism?
Mutation refers to a change in DNA sequence, while polymorphism refers to the presence of two or more variants in a population.
What does DNA sequencing determine?
The exact sequence of nucleotides within a DNA molecule.
Who developed the most common method of DNA sequencing?
Sanger and Coulson.
What is a single-stranded DNA (ssDNA) template required for?
Dideoxy DNA sequencing.
What is the role of dideoxynucleotides (ddNTPs) in DNA sequencing?
They cause chain termination during DNA synthesis.
What components are required for dideoxy DNA sequencing?
- Single-stranded DNA (ssDNA) template
- Short oligonucleotide primer
- DNA polymerase
- dNTPs
- Buffer
- MgCl2
What happens when a ddNTP is incorporated during DNA synthesis?
Synthesis stops because there is no 3’ OH group available for further elongation.
What are the four reactions in dideoxy DNA sequencing?
- A: ends with ddATP (opposite T)
- C: ends with ddCTP (opposite G)
- G: ends with ddGTP (opposite C)
- T: ends with ddTTP (opposite A)
What is the purpose of gel electrophoresis in DNA sequencing?
To separate DNA fragments from the four reactions.
What type of gel is commonly used for DNA sequencing?
Polyacrylamide gel matrix.
What is the typical composition of a standard sequencing gel?
6% acrylamide with a 19:1 ratio of acrylamide to bis-acrylamide.
What is the purpose of denaturing conditions in gel electrophoresis?
To ensure ssDNA fragments have no secondary structure for accurate size determination.
How are new strands visualized in DNA sequencing?
By labelling the primer or incorporating a label during synthesis.
What kind of labels were used in the past for DNA sequencing?
- 32P: high energy
- 33P: low energy
- 35S: low energy
What modern method is used to label dideoxynucleotides?
Fluorescent labels.
Why are termination products not labelled in modern sequencing?
Only strands terminated by dideoxy incorporation are labelled, reducing background signal.
What is the role of DNA polymerases in sequencing?
- High processivity
- Negligible 5’ to 3’ exonuclease activity
- Negligible 3’ to 5’ exonuclease activity
- Thermostable
What is thermocycle sequencing?
A method where new strand synthesis is repeated multiple times using a single primer and template.
What does the use of thermocycles and fluorescent labels allow in sequencing?
Automated Sequencing.
What are the characteristics of the four fluorescent labels used in modern sequencing?
Each emits energy at different wavelengths following excitation.
What is the Human Genome Project?
An international project that sequenced the first human genome from 1990 to 2003.
What was the cost of the Human Genome Project?
$2.7 billion.
What is the current price for whole genome sequencing (WGS)?
Less than $300.
