MOL BIO LAB L10 (Finals- Sequencing Methods) Flashcards
The first DNA sequencing method to be developed and is still widely used today.
Sanger sequencing
Two main types of DNA sequencing methods:
1) Sanger sequencing
2) Next-generation sequencing (NGS)
The process of determining the order of nucleotides in a DNA molecule.
DNA sequencing
First step of Sequencing by ligation (SBL)
DNA molecule is first fragmented into small pieces.
Variety of techniques of NGS
- sequencing by synthesis (SBS)
- sequencing by ligation (SBL)
This determines the genetic code
order of nucleotides in a DNA molecule
The most common type of NGS method
Sequencing by synthesis (SBS)
First step of Sequencing by synthesis (SBS)
The DNA molecule is first attached to a solid surface.
These methods have revolutionized the field of genomics, making it possible to sequence entire genomes at a relatively low cost
Next-generation sequencing (NGS)
NGS methods typically involve massively __________ the sequencing process
parallelizing
Arrange in order the following steps involved in Sequencing by synthesis:
____ A laser is used to image the surface and determine the order of the nucleotides in the DNA molecule.
____ A mixture of nucleotides is added to the surface.
____ The nucleotides will bind to the complementary nucleotides in the DNA molecule.
____ The DNA molecule is first attached to a solid surface.
__4__ A laser is used to image the surface and determine the order of the nucleotides in the
DNA molecule.
__2__ A mixture of nucleotides are added to the surface.
__3__ The nucleotides will bind to the complementary nucleotides in the DNA molecule.
__1__ The DNA molecule is attached to a solid surface.
This typically involve massively parallelizing the sequencing process, meaning that many DNA molecules are sequenced simultaneously.
Next-generation sequencing (NGS)
In SBL, this reads the order of the nucleotides in the chain.
Sequencing machine
In SBS, this is used to image the surface and determine the order of the nucleotides in the DNA molecule.
A laser
In SBL, the fragments are ________ together to form a long chain.
ligated
These are similar to nucleotides, but they lack a 3’-hydroxyl group
Dideoxynucleotides
To perform Sanger sequencing, a DNA sample is amplified using ______ and then incubated with a mixture of ______and ______
PCR; ddNTPs and dNTPs
In Sanger sequencing, the DNA fragments are separated by ____________ and the sequence of the DNA molecule is determined by ________ the order of the bands on the gel.
gel electrophoresis; reading
Arrange in order the following steps involved in Sequencing by ligation:
____ The fragments are attached to adapters.
____ The fragments are then ligated together to form a long chain.
____ DNA molecule is fragmented into small pieces.
____ The sequencing machine then reads the order of the nucleotides in the chain.
____ The adapters contain sequences that are known to bind to the sequencing machine.
__2__ The fragments are
attached to adapters.
__4__ The fragments are then ligated together to form a long chain.
__1__ DNA molecule is fragmented into small pieces.
__5__ The sequencing machine then reads the order of the nucleotides in the chain.
__3__ The adapters contain sequences that are known to bind to the sequencing machine.
Next-generation sequencing (NGS) methods revolutionized DNA sequencing by making it possible to?
sequence large DNA molecules much faster and more cheaply
In SBL, the fragments are
attached to?
adapters
It is a relatively simple and straightforward method, but it can be time-consuming and expensive to sequence large DNA molecules.
Sanger sequencing
NGS is now used in a wide range
of applications, including:
research, diagnostics, and personalized medicine
Contain sequences that are known to bind to the sequencing machine.
Adapters
A collection of methods that can sequence DNA much faster and more cheaply
Next-generation sequencing (NGS)
A chain-termination method that uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis.
Sanger sequencing
Works by sequencing the DNA molecule one fragment at a time.
Sequencing by ligation (SBL)
This randomly terminates DNA synthesis, resulting in a series of DNA fragments of different lengths.
ddNTPs
Many DNA molecules are sequenced simultaneously.
Next-generation sequencing (NGS) methods
It works by sequencing the DNA
molecule one nucleotide at a time.
Sequencing by synthesis (SBS)
This is necessary for DNA synthesis to continue
3’-hydroxyl group
The set of instructions for building and maintaining an organism
Genetic code
Enumerate the nucleotides
- adenine (A)
- thymine (T)
- guanine (G)
- cytosine (C)
Arrange in order the following steps involved in Sanger sequencing:
____ The ddNTPs will randomly terminate DNA synthesis, resulting in a series of DNA fragments of different lengths.
____ The order of the nucleotides in the DNA molecule is determined by reading the order of the bands on the gel.
____ The DNA sample is amplified using PCR.
____ The DNA fragments are separated by gel electrophoresis.
____ The amplified DNA is incubated with a mixture of ddNTPs and dNTPs.
__3__ The ddNTPs will randomly terminate DNA synthesis, resulting in a series of DNA fragments of different lengths.
__5__ The order of the nucleotides in the DNA molecule is determined by reading the order of the bands on the gel.
__1__ The DNA sample is amplified using PCR.
__4__ The DNA fragments are separated by gel electrophoresis.
__2__ The amplified DNA is incubated with a mixture of ddNTPs and dNTPs.
