DNA SEQUENCING Flashcards
(38 cards)
is the process of reading the nucleotides present in DNA: determining the precise order of nucleotides within a DNA Molecule.
DNA Sequencing
refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule.
DNA sequencing
The________ (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological
information that cells use to develop and operate.
sequence of the bases
Types of Sequencing Methods:
- Maxam-Gilbert Sequencing
- Sanger Sequencing
- Capillary Sequencing
- Whole Genome Sequencing
FIRST GENERATION SEQUENCING TECHNOLOGIES
- Maxam-Gilbert sequencing method
- Sanger Sequencing method
- Capillary sequencing method (CapSeq)
NOTE:_______ method uses the principle of Sanger Technique
CapSeq method
- MAXAM-GILBERT SEQUENCING METHOD
• Developed in 1977 ( _______ and _____)
• a _______method allowing to sequence dsDNA without previous in vivo cloning steps.
• Nitrogenous base-specific reactions are carried out to modify the Adenosine (A), Cytidine (C), Guanosine (G) and Thymidine (T) residues, allowing the chemical cleavage of the ssDNA at the 5’-P side of such positions.
Allan Maxam and Walter Gilbert
chemical-degradation
• Nitrogenous base-specific reactions are carried out to modify the Adenosine (A), Cytidine (C), Guanosine (G) and Thymidine (T) residues, allowing the chemical cleavage of the ssDNA at the 5’-P side of such positions.
MAXAM-GILBERT SEQUENCING METHOD
• Developed in 1977
• a chemical-degradation method allowing to sequence dsDNA without previous in vivo cloning steps.
MAXAM-GILBERT SEQUENCING METHOD
HOW TO
INTERPRET SEQUENCE in
MAXAM-GILBERT METHOD?
Read in what direction ??
5’ to 3’
• Enzyme-based termination of DNA
strand
• Became the popular method for sequencing.
SANGER SEQUENCING
• Developed in 1977
• Also known as “dideoxy chain termination method”
SANGER SEQUENCING
• a DNA sequencing technique developed by Frederick Sanger that uses dideoxynucleotides (ddNTPs) to determine the sequence of nucleotides in a DNA fragment
SANGER SEQUENCING
HISTORY
In 1977, Frederick Sanger, Allan Maxam, and Walter Gilbert pioneered_______.
The ___________ method uses a chemical method that involves selective degradation of bases.
The most widely used method for DNA sequencing is the________ or _______
DNA sequencing
Maxam and Gilbert sequencing
Sanger or “dideoxy” method,
• a DNA sequencing method that uses a long, thin capillary tube to separate DNA fragments by size, allowing for the determination of their sequence
Capillary Sequencing
• During 1990s, In_______ sequencing machines, DNA fragments are separated by size through a long, thin, acrylic-fibre capillary (instead of an electrophoresis gel, as with the Sanger method).
capillary
TOOLS YOU CAN VIEW YOUR CHROMATOGRAMS
•
is a popular desktop application that was developed by Geospiza, Inc. for viewing trace data from Sanger DNA Sequencing (scf or ab 1 file formats).
FinchTV chromatogram viewer
Compression Sequence
Problem:
Sequence appears as multiple overlying traces, not always throughout the entire sequence.
How to identify?
Sequence data begins to show multiple overlapping traces after a point in the sequence, although the sequencing signal remains strong.
CAUSE
DNA fragments of different size with the same electrophoretic mobility, ie fragments that migrate on top of each other during electrophoresis.
High Background/Noisy Sequencing Signal
Problem:
LowlPoor Signal-to-noise ratio in sequence data causing unreadable sequence data at points in the sequence.
How to identify?
Observation of small peaks underneath all peaks in a sequence read.
CAUSE
- Too little DNA template is added to sequencing reaction.
- Primer binding to the template is not very efficient.
- DNA template was not purified well enough prior to the sequencing reaction and is contaminated.
No Readable Sequence Data
Problem:
Lack of sequence data
How to Identify:
Sequencing trace is non-uniform.
Unincorporated Dye Terminator Peaks at the beginning of the sequence are out-of-scale.
Basecalls contain many ambiguous bases (“N’).
Cause:
- Primer not annealing to template (no priming site).
- No DNA template present in sequencing
reaction. - Incorrect cycle sequencing conditions.
- DNA template is of poor quality.
Contaminated Plasmid DNA Preparation
Problem:
Contaminated plasmid DNA preparation leads to overlapping peaks in the sequence data.
How to identify?
Sequence data is clear out to the end of the insert cloning site, at which point peaks underneath other peaks can often be seen.
Cause:
Picking more than one clone for a single plasmid preparation, or picking a single colony that contains two or more plasmids, each with different inserts.
The critical difference between Sanger sequencing and NGS is_______
sequencing volume
While the ________only sequences a single DNA fragment at a time,
_____is massively parallel, that is, sequencing millions of fragments simultaneously per run.
This process translates into sequencing hundreds to thousands of genes at one time.
Sanger method
NGS
