What is DNA sequencing?

DNA Sequencing is determining the precise order of nucleotides in a piece of DNA

Why is it done?

DNA sequence is useful in studying fundamental biological processes and in applied fields such as
diagnostic or forensic research

WATCH AND MAKE NOTES:

https://www.youtube.com/watch?v=ONGdehkB8jU

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 research workers to find out the structure
of gene products.

BEFORE WE GO ANY FURTHER, let us recall the structure of a nucleotide.

NOW recall that nucleotides in a DNA strand are linked to each other by phosphor diester bonds
Two main methods are widely known to be used to sequence DNA:

1. The Chemical Method (also called the Maxam–Gilbert method after its inventors).

2. The Chain Termination Method (also known as the Sanger dideoxy method after its
inventor).

 Maxam–Gilbert technique depends on the relative chemical liability of different nucleotide

bonds, whereas the Sanger method interrupts elongation of DNA sequences by
incorporating dideoxynucleotides into the sequences.

 The chain termination method is the method more usually used because of its speed and
simplicity.

SANGER’S METHOD:

Sanger’s chain terminator method is more efficient and uses fewer toxic chemicals and lower
amount of radioactivity than the method of Maxam and Gilbert.

 The key principle of the Sanger method was the use of dideoxynucleotide triphosphates
(ddNTPs) as DNA chain terminators.

 The chain termination method requires a single-stranded DNA template, a DNA primer, a
DNA polymerase, radioactively or fluorescently labelled nucleotides, and modified
nucleotides that terminate DNA strand elongation.

 The DNA sample is divided into four separate sequencing reactions, containing all four of the
standard deoxynucleotides (dATP, dGTP, dCTP, dTTP) and the DNA polymerase.

 To each reaction is added only one of the four dideoxynucleotide (ddATP, ddGTP, ddCTP,
ddTTP) which are the chain terminating nucleotides, lacking a 3’-OH group required for the
formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand
extension and resulting in DNA fragments of varying length.

 The newly synthesized and labelled DNA fragments are heat denatured, and separated by
size by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four
reactions run in one of the four individual lanes (lanes A, T, G, C).

 The DNA bands are then visualized by autoradiography or UV light, and the DNA sequence
can be directly read off the X-ray film or gel image.

 A dark band in a lane indicates a DNA fragment that is result of chain termination after
incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP).

 The relative position of the different bands among the four lanes are then used to read
(from bottom to top) the DNA sequence.

 The technical variations of chain termination sequencing include tagging with nucleotides
containing radioactive phosphorus for labelling, or using a primer labelled at the 5’ end with
a fluorescent dye.

 Dye- primer sequencing facilitates reading in an optical system for faster and more
economical analysis and automation.
Key Features

 Uses dideoxy nucleotides to terminate DNA synthesis.

 DNA synthesis reactions in four separate tubes

 Radioactive dATP is also included in all the tubes so the DNA products will be radioactive.

 Yielding a series of DNA fragments whose sizes can be measured by electrophoresis.

 The last base in each of these fragments is known.

Advantage

Chain termination methods have greatly simplified DNA sequencing.

Limitations

 Non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA
sequence.

 DNA secondary structures affecting the fidelity of the sequence.

 Costly, time consuming, labour intense

WATCH https://www.youtube.com/watch?v=vK-HlMaitnE

GILBERT METHOD: In a nut shell---

 In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based
on chemical modification of DNA and subsequent cleavage at specific bases.

 The method requires radioactive labelling at one end and purification of the DNA fragment
to be sequenced.

 Chemical treatment generates breaks at a small proportions of one or two of the four
nucleotide based in each of four reactions (G,A+G, C, C+T).

 Thus a series of labelled fragments is generated, from the radiolabelled end to the first ‘cut’
site in each molecule.

 The fragments in the four reactions are arranged side by side in gel electrophoresis for size
separation.

 To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a
series of dark bands each corresponding to a radiolabelled DNA fragment, from which the
sequence may be inferred.

Key Features

 Base-specific cleavage of DNA by certain chemicals

 Four different chemicals, one for each base

  A set of DNA fragments of different sizes

 DNA fragments contain up to 500 nucleotides

Advantages

 Purified DNA can be read directly

 Homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences

 Can be used to analyze DNA protein interactions (i.e. footprinting)

 Can be used to analyze nucleic acid structure and epigenetic modifications to DNA

Disadvantages

 It requires extensive use of hazardous chemicals.

 It has a relatively complex set up / technical complexity.

 It is difficult to “scale up” and cannot be used to analyze more than 500 base pairs.

 The read length decreases from incomplete cleavage reactions.

 It is difficult to make Maxam-Gilbert sequencing based DNA kits.

WATCH and make notes https://www.youtube.com/watch?v=_B5Dj8PL4E0

Test your ddNTP reading comprehension with these practice questions:

Q 1. A scientist wants to determine the sequence of a gene associated with a genetic disease. She
takes samples of DNA a person who has the disease-causing version of the gene them into four
separate test tubes, each containing nucleotides, DNA polymerase, and primers.

Then, into each tube, she adds one type of dideoxynucleotide — ddATP, ddGTP, ddCTP, or ddTTP —
so that each tube will produce DNA fragments that end with a particular nucleotide (A, G, C, or T).
After running the sequencing reactions, she loads the results of each tube into a gel and separates
the fragments using gel electrophoresis.

Based on the results in her gel, shown in this figure, what is the sequence of the gene?
 A. DNA travels toward the positive electrode in a gel, and the smallest fragments move the
greatest distance, so the bands in the gel that are closest to the + are the smallest bands.
The shortest fragment (the lowest band) is in the lane that was loaded with ddCTPs, so the
first nucleotide in the DNA strand must be C. The next band is in the lane that was loaded
with ddTTPs, so the next nucleotide must be T. The next band is again in the C lane, so the
next nucleotide must be a C. If you continue reading the gel from the + side to the – side —
in other words, from the bottom to the top — you can figure out the rest of the DNA
sequence. The entire sequence is CTC AGC CAT AGG.

Q 2. A young woman whose mother died from early onset Alzheimer’s wants to know whether she is
at risk for developing the disease. The disease is caused by a dominant allele, so if the woman has
just a single copy of the disease-causing allele, she’ll develop the disease.

Describe how a scientist could use a blood sample from the young woman to screen her for the
presence of the disease-causing allele. Be sure to include all the necessary techniques, starting with
the blood sample.

A. The scientist would extract DNA from the sample of blood and then use either DNA sequencing
or restriction enzymes to determine whether the young woman carries the disease-causing
allele.

DNA sequencing method: The scientist reads the sequences of the young woman’s alleles and
compares them to the known sequences of the normal and disease alleles. The scientist separates
the young woman’s DNA sample and places it into four tubes. Each tube contains primers for the
Alzheimer’s gene and lots of nucleotides.

One of each tube contains a different fluorescently labeled ddNTP: one with ddATP, one with ddCTP,
one with ddGTP, and one with ddTTP. The scientist places the tubes into a cycle sequencer, where
they go through many rounds of DNA replication.
Then the scientist runs the samples through a gel that separates them by size. The computer detects
the fluorescent signal at the end of each piece of DNA from smallest to largest and uses it to
reconstruct the DNA sequences for the young woman’s alleles.

Restriction enzyme method: The scientist uses PCR on the DNA from the blood to make many copies
of the gene associated with Alzheimer’s. Then the scientist uses a restriction enzyme that’s known to
cut differently within the normal and disease forms of the gene. The scientist cuts the young
woman’s DNA sample with the restriction enzyme and then separates the DNA using gel
electrophoresis. The scientist compares the sizes of the DNA fragments produced by the young
woman’s DNA to the known pattern generated by the normal and disease alleles.