GENE MUTATIONS AND MUTATION DETECTION

GENE MUTATIONS

Also known as “Gene Variant”

GENE MUTATIONS

Permanent change in DNA sequence that makes up a gene

Gene Variant

It is formerly called “Gene Mutation”, but changes in DNA does not always cause disease so the accurate term is

Radiation

UV lights

Carcinogens

Environmental Factors that cause mutation

Deletions

Insertions

Inversions

Translocations

Other factors that affect base pairing in a gene

Point Mutations

alterations of a single or a few base pairs/ single base change

GENE MUTATIONS

It includes deletions, insertions, inversions, translocations, and other changes that can affect base pairing in a gene

Point Mutations

are increasingly analyzed by sequencing methods

Sequencing

not only directly detects the mutated base or bases but also provides the context of neighboring bases

Silent

Conservative

Non-conservative

Non-sense

Frameshift

Types of Gene Mutations

Silent Gene Mutation

Substitution of one nucleotide with a different nucleotide but it doesn’t change the amino acid sequence

- Example: GCC → GCA (both produces alanine even if point mutation happened)

Conservative Gene Mutation

may change the amino acid sequence, but the replacement and the original amino acid have similar biochemical properties

Conservative Gene Mutation

Leucine and Valine have the same function and biochemical characteristics.

- GTG (Leu)→ GUG (Val) = no effect in the sequence

Non-conservative Gene Mutation

replacement of an amino acid with a biochemically different amino acid

- Proline → Glutamine

Non-sense Gene Mutation

Terminates proteins prematurely when a nucleotide substitution produces a stop codon instead of an amino acid codon

- 3 STOP CODON: UAA, UAG, UGA

Frameshift Gene Mutation

Insertion or deletion of other than a multiple of three Nucleotides

- Throwing the triplet code out of the frame

Nonconservative, Nonsense, Frameshift

these mutations will generate different phenotypes, depending on where they occur along the protein sequence

Point Mutation

in the end of a coding region may have minimal consequences, whereas mutations at the beginning of a coding sequence are more likely to result in drastic alterations or even effective deletion of the protein coding region.

NONE

DNA Sequence: ATG CAG GTG ACC TCA GTG

Amino Acid Sequence: M Q V T S V

Type of Mutation:??

SILENT

DNA Sequence: ATG CAG GTT ACC TCA GTG

Amino Acid Sequence: M Q V T S V

Type of Mutation:??

CONSERVATIVE

DNA Sequence: ATG CAA GTG ACC TCA GTG

Amino Acid Sequence: M Q L T S V

Type of Mutation: ??

NONCONSERVATIVE

DNA Sequence: ATG CCG GTG ACC TCA GTG

Amino Acid Sequence: M P V T S V

Type of Mutation:??

NONSENSE

DNA Sequence: ATG CAG GTG ACC TGA GTG

Amino Acid Sequence: M Q V T ter

Type of Mutation: ??

FRAMESHIFT

DNA Sequence: ATG CAG GTG AAC CTC AGT G

Amino Acid Sequence: M Q V NLS

Type of Mutation:??

BIOCHEMICAL METHODS

Used to detect of quantify the altered protein product

BIOCHEMICAL METHODS

Product: PROTEIN

BIOCHEMICAL METHODS

If you have problem in the sequence of DNA, that will affect the amino acid sequence→ If AA sequence is altered → protein product will be altered → Phenotype of organism is changed

BIOCHEMICAL METHODS

Used to directly analyze (more direct analysis) the change in protein structure or function rather than to search for potential point mutations

BIOCHEMICAL METHODS

Detects metabolic defects and protein or amino acid alterations

Immunoassays

Immunohistochemistry

Commonly used biochemical methods

Immunoassays

if you want to check metabolites in sample

Immunohistochemistry

detect protein abnormalities in-situ (inside cell) so that the tissue and intracellular location of mutant proteins can be observed, in-place detected

High-Performance liquid or Gas Chromatography (HPLC/GC)

Mass Spectrometry

Automated, frequently used:

ENZYME IMMUNOASSAYS

formats for direct antigen detection

ENZYME IMMUNOASSAYS

Involve the use of specific antibodies or other ligands to detect the presence of the target molecules

ENZYME IMMUNOASSAYS

Utilizes plate wells, strips, or capillaries coated with capture antibody or antigen depending on what you are detecting

ENZYME IMMUNOASSAYS

If the analyte is present in the test fluid, it will bind to the known substances that are bound to your plate or well ex. immobilized antibody - Antigen or antibody specific

ENZYME IMMUNOASSAYS

Detection is by the use of a secondary antibody conjugated to enzyme

Secondary Antibody

- highly specific to the analyte

- If there is an analyte, there will be a signal because the enzyme will be activated once the substrate is added. It will produce chemiluminescence, fluorescence or color signals depending on the substrate added.

ANTIGEN DETECTION

Uses immobilized antibody conjugated with enzyme e.g AP & HPO

ANTIBODY DETECTION

Uses immobilized antigen and antihuman antibody conjugates

ANTIBODY DETECTION

Detect presence of ANTIBODY

ANTIGEN

Immobilized particle

Secondary antibody

Anti-immunoglobulin

IMMUNOHISTOCHEMISTRY

Performed on thin (<5 micron) slices of fixed or 5- to 15 micron slices of frozen tissue

IMMUNOHISTOCHEMISTRY

It provides the advantage of integrating target detection, localization, and quantification in the context of tissue morphology.

IMMUNOHISTOCHEMISTRY

Imaging or microscopic observation of antibody binding → fluorescent or colorimetric

IMMUNOHISTOCHEMISTRY

Imaging or microscopic observation of antibody binding → fluorescent or colorimetric

IMMUNOHISTOCHEMISTRY

You have to observe the antibody that binds to the changes on the cell.

fluorescent dyes or signals

If you don't have enzymes, _____ can be used.

Cy5

Phycoerythrin

these are examples of fluorescence producing signals that can be used instead of enzymes.

red or brown

The color generation depends on the substrate that you have. Most of the time, it produces _______ immunohistochemistry staining.

brown

Most of the time, the color of immunohistochemistry is ____?

there are changes in the protein sequence in the tissue

If you are performing immunohistochemistry, Check for the brownish color to be produced under the microscope. Which indicates that ______?

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

is the basis for separation/analysis instruments such as amino acid analyzers

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Check for the migration of the molecules in the machine.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

It is an automated method. You have to inject the sample in the machine and the movement/ migration of the molecules will be detected by the machine.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Most of the time, this is the method on how we do the amino acid analyzers

Mobile Phase

Stationary phase

two phases of HPLC

Mobile Phase HPLC

just like with other chromatographic assays; a liquid.

Higher powered ultra-HPLC (UHPLC) columns

columns that have been devised to increase resolution and lower separation time while using less solvent

GAS CHROMATOGRAPHY (GC)

It is an automated method of analysis, the mobile phase is an inert gas, and the stationary phase is a high-boiling point liquid that is absorbed to an inert solid support in the column.

GAS CHROMATOGRAPHY (GC)

The sample is introduced to the column and vaporized into a gas. The inert gas carries the sample through the column.

GAS CHROMATOGRAPHY (GC)

Alternatively, this may be coupled to a mass spectrometer.

GAS CHROMATOGRAPHY (GC)

is used for detection of drugs and poisons and their metabolites in biological samples.

GAS CHROMATOGRAPHY (GC)

Coupled with MS, ____ is being used to detect biomarkers of disease.

GAS- LIQUID CHROMATOGRAPHY

is the separation of vaporized sample through a column of inert carrier gas and liquid stationary phase that differentially adsorbs molecules.

MASS SPECTROMETRY

Automated method that converts molecules to ions that can be moved in a magnetic field based on their charge and mass

MASS SPECTROMETRY

it relies on the ionization or the conversion of the molecules into ions

MASS SPECTROMETRY

The heavier the molecules are, their movement will be affected.

MASS SPECTROMETRY

heavy/high mass, the migration will be slower.

Primers

are bound to test DNA template just adjacent (5ʹ) to the base position to be analyzed

Primers

Used to check nucleic acid changes

Primers

need to check for single base changes or point mutations.

Primers

are bound to test DNA template just adjacent (5ʹ) to the base position to be analyzed

• The ____ will be extended with the dNTP

dideoxynucleotide (ddNTP)

Extension and termination with a ____?

dNTP

will be added and the synthesis of DNA will stop upon incorporation of the ddNTP

Extension and termination with a dideoxynucleotide (ddNTP)

it will terminate the extension in one base and changes the mass and charge of the primer

Extension and termination with a dideoxynucleotide (ddNTP)

the sizes of the product DNA will change hence it will change the mass and charge ratio of the extended primer

MASS SPECTROMETER

converts molecules to ions that can be moved in a magnetic field based on their charge and mass.

MASS SPECTROMETRY

In this automated method, an ion source sends high energy electrons that hit the target sample molecules, separating them into ions, usually cations with the loss of one or two electrons.

Classical Molecular Methodology

Mutation detection by analysis of nucleic acids is considered the _____ ???

Mutation Detection

is more specific when performing molecular test

Somatic Mutation

occur in a single body cell and cannot be inherited

- The changes in the sequence might be induced by exposure to carcinogens (environmental factors)

Germline Mutation

occur in gametes and can be passed onto offspring - being inherited through generations

Hybridization- based methods

Sequence (polymerization)-based methods

Enzymatic or chemical cleavage methods

Sequence detection methods can be generally classified according to three broad approaches: