DNA sequencing

Hello , my name is Livija and today I want to talk about DNA sequencing. In my
opinion, firstly we need to find out what it is. So, DNA sequencing refers to the
general laboratory technique for determining the exact sequence of nucleotides,
or bases, in a DNA molecule. Most people may wonder why DNA sequencing is
needed. The sequence of DNA can reveal lots of genetic information, helping
identify genes that code for proteins, as well as mutations that can cause disease,
also are yielding information about the evolution of genomes; the location of
coding regions. In 1977, Frederick Sanger, Allan Maxam, and Walter Gilbert
pioneered DNA sequencing. The Maxam and Gilbert sequencing method uses a
chemical method. The most widely used method for DNA sequencing is the
Sanger method. In this method, single-stranded DNA is mixed with a radioactively
labeled primer to provide the 3-OH required for DNA polymerase to initiate DNA
synthesis. Four DNA synthesis reaction mixes are set up using template The
radioactive products of each reaction mixture are separated by polyacrylamide
gel electrophoresis and located by exposing to X-ray film. The nucleotide
sequence of the newly synthesized DNA is read directly from the autoradiogram
beginning at the bottom of the autoradiogram. The sequence in the original
template strand is its complement (3 → 5 ). The random incorporation of
dideoxy ATP into the growing chain generates a series of smaller DNA fragments
ending at all possible positions where adenine is found in the newly synthesized
fragments. An exposed X-ray film of a DNA sequencing gel. The four lanes
represent A, C, G, and T dideoxy reaction mixes, respectively. Now I want to talk
about automated DNA sequencing. In 1986, Leroy Hood and Lloyd Smith
automated Sanger’s method. In this new sequencing technology, radioactive
markers are replaced with fluorescent ones. Each ddNTP terminator is tagged
with a different color of fluorophore: red, green, blue, or yellow. In this system
each automated sequencer was able to produce 4800 bases of sequence. The
diagram depicts the steps involved in automated DNA sequencing. the primers
used in each of
the four reactions are tagged with different fluorescent molecules. The products
from each tube will emit a different-colored fluorescence when excited by light. In
my opinion automated DNA sequencing have a lot of benefits because In addition
to better process control, higher data quality, and accurate and efficient work
flows, automated sample preparation for DNA sequencing can bring a level of
repeatability between-run and between-technicians difficult to achieve by even
the most experienced and diligent of laboratories. However, as with any
invention, automated dna sequencing has certain disadvantages: Instruments cost
are high, a highly trained personnel can only operate. We discussed about Sanger
method , automated DNA sequencing and now I want to talk about next-
generation sequencing. The basic next-generation sequencing process involves
fragmenting DNA/RNA into multiple pieces, adding adapters, sequencing the
libraries, and reassembling them to form a genomic sequence. So, DNA sequence
information is important for scientists investigating the functions of genes.
Understanding the sequences of DNA can be applied in various settings like: DNA
sequencing now forms the base of biologic research and is applied in
biotechnology, forensic biology, virology, and medical diagnoses.