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DNA Sequencing

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20 views7 pages

DNA Sequencing

Uploaded by

Somiyyah
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA Sequencing

 DNA sequencing refers to methods for determining the order


of the nucleotides bases adenine, guanine, cytosine and
thymine in a molecule of DNA

 Chain Termination Method (also known as the Sanger dideoxy


method after its inventor).

 Chemical Method (also called the Maxam–Gilbert method after


its inventors).

 Next Generation sequencing (NGS)


Significance of DNA Sequencing

• Information obtained by DNA sequencing makes it


possible to understand or alter the function of genes.

• DNA sequence analysis demonstrates regulatory


regions that control gene expression and genetic
“hot spots” particularly susceptible to mutation.

• Comparison of DNA sequences shows evolutionary


relationships that provide a framework for definite
classification of microorganisms including viruses.
• Comparison of DNA sequences facilitates identification
of conserved regions, which are useful for development of
specific hybridization probes to detect microorganisms
including viruses in clinical samples.

• DNA sequencing has become sufficiently fast and


inexpensive to allow laboratory determination of
microbial sequences for identification of microbes.

• Sequencing of the 16S ribosomal subunit can be used to


identify specific bacteria.

• Sequencing of viruses can be used to identify the virus


and distinguish different strains.

• DNA sequencing shows gene structure that helps


Sanger dideoxy method

 Single-stranded DNA (ssDNA) to be sequenced is used as the template


for DNA synthesis by DNA polymerase.

 A radiolabeled primer complementary to the 3ʹ-end of the target DNA is


added, along with the four deoxyribonucleoside triphosphates (dNTPs:
dATP, dCTTP, dGTP, dCTTP).

 The sample is divided into four reaction tubes, and a small amount of
one of the four dideoxyribonucleoside triphosphates (ddNTPs) is added
to each tube. Because it contains no 3ʹ-hydroxyl group, incorporation of
a ddNMP terminates elongation at that point.

 The products of this reaction consist of a mixture of DNA strands of


different lengths, each terminating at a specific base. They can be
Separated by gel electrophoresis, followed by autoradiography, yields
a pattern of bands from which the DNA base sequence can be read
Separation of the
various DNA
products by size
using
polyacrylamide gel
electrophoresis,
followed by
autoradiography,
yields a pattern of
bands from which
the DNA base
sequence can be
read.

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