Subject Name: Genetic Engineering-II

Subject Code: MTI-605

Unit No:5
Unit Name: Molecular Diagnostics
Faculty Name : Dr. V. Suresh Gupta

Sub Topic Name NASBA

NASBA

SEQUENCE BASED DETECTION

NASBA

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What is DNA sequencing?

Knowledge of DNA sequences has become indispensable for basic
biological research, and in numerous applied fields such as diagnostic,
biotechnology, forensic biology, and biological systematic.
DNA sequencing is the process of determining the precise order of
nucleotides within a DNA molecule.
It includes any method or technology that is used to determine the
order of the four basesAdenine, Guanine, Cytosine, and Thymine
in a strand of DNA.
The first DNA sequences were obtained in the early 1970s by
academic researchers using laborious methods based on twodimensional chromatography.

NASBA

Inventory Antiquity
Beginning in the early 1970's, Frederick Sanger and his colleagues of
the University of Cambridge founded a series of methods called
Chain Termination methods.
It is based on the selective incorporation of chainterminating dideoxynucleotides by DNA polymerase during in
vitro DNA replication.
They were quickly replaced in popularity by the Maxam-Gilbert DNA
sequencing method, which was invented by Allan Maxam and Walter
Gilbert around '76 - '77
It is based on first chemically modifying DNA and then cleaving it at
specific bases.

NASBA

What is Sequence Based Detection?

Advances in molecular biology have led to the development of more
sensitive, more specific and more rapid testing procedures based on
nucleic acid analysis.
These techniques enable the amplification of small quantities of specific
nucleic acid target sequences to concentrations well above the detection
limit of many conventional detection methods.
Hence they have become powerful investigation tools for scientists from a
wide range of disciplines in the research field.
It is used to apply genetic testing to the diagnosis, management, and
genetic counseling of patients and families with specific inherited
conditions.

NASBA

NASBA- Nucleic Acid Sequence-Based Amplification

RNA detection is commonly done using RT-PCR, a time consuming
process often resulting in false positives due to cross contamination.
Alternatively, nucleic acid sequence-based amplification (NASBA) is a
one step isothermal process (usually at a constant temperature of 41C)
for amplifying RNA and does not provide false positive results.
NASBA has proven to be successful in detection of both viral and
bacterial RNA in clinical samples.
A NASBA reaction consists of avian myeloblastosis virus (AMV)
reverse transcriptase (RT), T7 RNA polymerase and RNase H with
two oligonucleotide primers. The amplification is more than 1012 fold
in 90 to 120 minutes.

NASBA

NASBA Protocol

NASBA
This mechanism requires two short single stranded DNA fragments primers and
three enzymes.
1.
2.

3.
4.

5.

The viral RNA strands are represented as the sense strand present in the
original samples.
Primer P1 binds to the RNA and is elongated by reverse transcriptase (AMVRT). The RNA strand of the yielded DNA: RNA hybrid is hydrolyzed by
RNase H.
After the binding of P1, primer P2 can also bind. Primer P2 is then elongated
by AMV RT, yielding a double-stranded DNA molecule.
Primer P1 is designed in such a manner that when it forms a doublestranded DNA, it codes for a T7 RNA polymerase Promoter site. This helps in
generating antisense RNA copies using a DNA template.
The new copies of DNA are generated using RNA. The process is same as
followed for sense strand. Here in this case, P2 will bind first.

NASBA
In a NASBA reaction, DNA is the final product formed. It has a promoter
region. This allows T7 RNA polymerase to use it as a template.
The reaction sequences starting from the sense and antisense RNA
strands are different but they are considered to be kinetically identical.
They both are referred to as cDNA.

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NASBA vs. RT-PCR

NASBA needs shorter
assay time.
Availability of reagents
allows the method to be
used
for
frequent
diagnostic usage.
It is an isothermal process
running at 41C.

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RT-PCR has a longer

assay period.
Limited availability of
reagents make the
technique
usage
restricted.
Requires thermocycling
steps in procedures.

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Application of the NASBA

1. Retroviruses are being detected and quantified by only RNA
genome amplification and not the proviral DNA copies.
2. It can measure replication of DNA viruses by detecting late mRNA
expression.
3. It supports the detection of human mRNA sequences without the
risk of DNA contamination.
4. Gene expression studies can be done without intron flanking
primers or DNAses.
5. Mapping of recombination hotspots using sequencing-based
detection of ssDNA.
6. Detection of medically important Candida species.

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Thank You