Gene Mutations-Gene mutations include deletions,
insertions, inversions, translocations, and other changes
that can affect one base pair to hundreds or thousands
of base pairs.
Types of Gene Mutations
1.Silent-Substitution of one nucleotide with a different
nucleotide may be silent; that is, without changing the
amino acid sequence
2.Conservative substitutions -may change the amino
acid sequence, but the replacement and the original
amino acid have similar biochemical properties, e.g.,
leucine for valine, and the change will not affect protein
function significantly.
3.nonconservative -mutation is the substitution of a
biochemically different amino acid, e.g., proline for
glutamine, which changes the biochemical nature of the
protein
4.nonsense mutation -terminates proteins prematurely
when a nucleotide substitution produces a stop codon
instead of an amino acid codon
5.frameshift mutation -Insertion or deletion of more or
fewer than three nucleotides results in a frameshift
mutation, throwing the triplet code out of frame
Detection of Gene Mutations- Sequence detection
methods can be generally classified according to three
broad approaches: hybridizationbased methods,
sequence (polymerization)-based methods, and
enzymatic or chemical cleavage methods.
1.Hybridization-Based Methods
Single-Strand Conformation Polymorphism The
method is based on the preference of DNA (as well as
RNA) to exist in a double-stranded, rather than single-
stranded, state. In the absence of a complementary
strand, nucleic acids form intrastrand duplexes to attain
as much of a doublestranded condition as possible.
Conformer-the shape of which is determined by the
primary sequence of the folded strand.
Denaturing Gradient Gel Electrophoresis Denaturing
gradient gel electrophoresis (DGGE) -exploits
differences in denaturation between a normal and
mutated DNA molecule caused by even one nucleotide
difference in a sequence.
-Two methods that are similar in design to DGGE are
constant gradient gel electrophoresis (CDGE18,19) and
temporal temperature gradient gel electrophoresis
(TTGE20
Allele-Specific Oligomer Hybridization Allele-specific
hybridization, or allele specific oligomer hybridization
(ASO), utilizes the differences in melting temperatures
of short sequences of ~20 bases with one or two
mismatches and those with no mismatches
ASO is a dot blot method, similar to Southern blot using
immobilized target and labeled probe in solution. It has
been used to test for known, frequently occurring
mutations; for example, in the BRCA1 and BRCA2 gene
mutations frequently observed in inherited breast
cancer30 and the p16 gene mutations in familial
melanoma
Melt Curve Analysis -Like DGGE and related methods,
melt curve analysis (MCA) exploits the sequence- and
stacking-directed denaturation characteristics of DNA
duplexes.
Inversion Probe Assay The molecular inversion probe
system was designed as a method for detection of SNPs
in DNA.46 The molecular inversion probe is a linear
probe containing two specific regions, one at each end;
primer binding sites; and a 20 nucleotide–long unique
sequence tag
Heteroduplex Analysis- Solution hybridization and
electrophoresis of test amplicons mixed with reference
amplicons can reveal mutations. To form
heteroduplexes, nonidentical doublestranded DNA
duplexes are heated to a temperature that results in
complete denaturation of the double-stranded DNA
Conformation-sensitive gel electrophoresis- is a
heteroduplex analysis method in which the
heteroduplexes are resolved on a 1,4-bis (acrolyl)
piperazine gel with ethylene glycol and formamide as target site or changes the size of a fragment generated
mildly denaturing solvents to optimize conformational by a restriction enzyme, restriction fragment length
differences polymorphism (RFLP) analysis can be used to detect the
sequence alteration
standard tiling-In this format, the base substitution in
the immobilized probe is always in the twelfth position - To perform PCR-RFLP, the region surrounding the
from its 3′ end. Commonly occurring mutations can be mutation is amplified, and the mutation is detected by
targeted in another type of format cutting the amplicon with the appropriate restriction
enzyme
redundant tiling - in which the same mutation is placed
at different positions in the probe (at the 5′ end, in the Heteroduplex Analysis With Single-Strand Specific
middle, or at the 3′ end). Nucleases -The detection sensitivity of heteroduplex
analysis can be increased by using single-strand–specific
Bead array technology-utilizes sets of color-coded
nucleases, e.g., S1 nuclease, that cleave heteroduplexes
polystyrene beads in suspension as the solid matrix. In
at the mispaired bases.
an extension of the FlowMetrix system,61 100 sets of
beads are dyed with distinct fluorochrome mixes Base excision sequence scanning (BESS) -is a PCR
amplification in the presence of small amounts of
2. Sequencing (Polymerization)- Based Methods
deoxyuridine triphosphate (dUTP) added to the reaction
Sequence-specific PCR (SSP-PCR)- is commonly used to mix, followed by treatment with excision enzymes that
detect point mutations and other single nucleotide cleave the fragment at the dU sites.
polymorphisms. There are numerous modifications to
Nonisotopic RNase cleavage assay (NIRCA)- is a
the method, which involves careful design of primers
heteroduplex analysis using duplex RNA.103 The
such that the primer 3′ end falls on the nucleotide to be
sequences to be scanned are amplified using primers
analyzed. Unlike the 5′ end, the 3′ end of a primer must
tailed with promoter sequences of 20–25 bp.
match the template perfectly to be extended by Taq
polymerase Invader- is a method developed by Third Wave
Technologies that does not require PCR amplification of
Allelic Discrimination With Fluorogenic Probes -
samples.107–109 Premixed reagents are added to a
Thermal cyclers with fluorescent detection support
standard 96 well plate along with the test specimens
allelic discrimination with fluorogenic probes. This
and controls.
method is an extension of the 5′ nuclease PCR assay
using two probes labeled with different fluors Chemical cleavage of mismatches (CCM)-exploits the
susceptibility of specific base mismatches to
Dideoxy DNA fingerprinting (ddF) is a modified chain
modification by different chemicals.114 Mismatched
termination sequencing procedure .For this analysis, a
cytosines and thymines are modified by hydroxylamine
single dideoxynucleotide is used to generate a series of
and osmium tetroxide, respectively.
terminated fragments that are resolved in one lane of a
nondenaturing polyacrylamide gel. Gene Mutation Nomenclature- Accurate testing and
reporting of gene mutations require a descriptive and
Protein Truncation Test Nonsense or frameshift
consistent system of expressing mutations and
mutations -cause premature truncation of proteins. The
polymorphisms.
protein truncation test (also called in vitro synthesized
protein or in vitro transcription/translation) is designed
to detect truncated proteins as an indication of the
presence of DNA mutations.This procedure uses a PCR
product containing the area of the gene likely to have a
truncating DNA mutation

3.Cleavage Methods

Restriction Fragment Length Polymorphisms-If a

mutation changes the structure of a restriction enzyme

DNA Sequencing- In the clinical laboratory, DNA
sequence information (the order of nucleotides in the
DNA molecule) is used routinely for a variety of
purposes, including detecting mutations, typing
microorganisms, identifying human haplotypes, and
designating polymorphisms
A.Direct Sequencing -The importance of knowing the
order, or sequence, of nucleotides on the DNA chain
was appreciated in the earliest days of molecular
analysis. Elegant genetic experiments with
microorganisms detected molecular changes indirectly
at the nucleotide level.
- Direct determination of the nucleotide sequence, or
DNA sequencing, is the most definitive molecular
method to identify genetic lesions.
1.Manual Sequencing
The chemistry for automated sequencing is the same as
described for manual sequencing, using double-
stranded templates and cycle sequencing.
Approaches to Automated Sequencing
There are two approaches to automated fluorescent
sequencing: dye primer and dye terminator
sequencing . The goal of both approaches is the same:
to label the fragments synthesized during the
sequencing reaction according to their terminal ddNTP.
Electrophoresis and Sequence Interpretation -Both dye
primer and dye terminator sequencing reactions are
loaded onto a slab gel or capillary gel in a single lane.
The fluorescent dye colors, rather than lane assignment,
distinguish which nucleotide is at the end of each
fragment.
- Fluorescent detection equipment yields results as an
electropherogram, rather than a gel pattern.

Direct determination of the order, or sequence, of B.Pyrosequencing Chain termination sequencing is the
nucleotides in a DNA polymer is the most specific and most widely used method to determine DNA sequence.
direct method for identifying genetic lesions Other methods have been developed that yield the
(mutations) or polymorphisms, especially when looking same information but not with the throughput capacity
for changes affecting only one or two nucleotides. of the chain termination method.

- Two types of sequencing methods have been used

most extensively: the Maxam-Gilbert method2 and the
Sanger method.

The Maxam-Gilbert chemical sequencing method was

developed in the late 1970s by Allan M. Maxam and
Walter Gilbert. Maxam-Gilbert sequencing requires a
double- or single-stranded version of the DNA region to
be sequenced, with one end radioactively labeled.

Dideoxy (Sanger) Sequencing -The original dideoxy

chain termination sequencing methods required a
single-stranded template. Templates up to a few
thousand bases long could be produced using M13
bacteriophage, a bacterial virus with a singlestranded
DNA genome.

- This virus replicates by infecting Escherichia coli, in

which the viral single-stranded circular genome is
C.Bisulfite DNA sequencing, or methylation-specific
converted to a double-stranded plasmid, called the
sequencing, is a modification of chain termination
replication factor (RF).
sequencing designed to detect methylated
2. Automated Fluorescent Sequencing nucleotides.12,13 Methylation of cytosine residues in
DNA is an important part of regulation of gene
expression and chromatin structure
D.Bioinformatics -is the merger of biology with
information technology. Part of the practice in this field
is biological analysis in silico; that is, by computer rather
than in the laboratory.

E.The Human Genome Project From the first

description of its double helical structure in 1953 to the
creation of the first recombinant molecule in the
laboratory in 1972, DNA and the chemical nature of the
arrangement of its nucleotides have attracted interest.
Gradually, this information began to accumulate, first
regarding simple microorganisms and then partially in
lower and higher eukaryotes.