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Gene Mutation and Mutation Detection Technique

Gene Mutation and Mutation Detection Technique
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College of Medicine

291 Documents
Students shared 291 documents in this course
Academic year: 2022/2023
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La Consolacion University Philippines

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Gene Mutation and Mutation

Detection Technique

  • AKA “Gene Variant”.
  • A permanent change in a DNA sequence.
  • Mutations arise spontaneously at low frequency owing to the chemical instability of the purine and pyramidine bases.
  1. Mutagenic
  • Overexpose to ultraviolet light.
  1. Chemical carcinogens
  • Eg. Aflatoxin B1 can cause mutation.

Gene Mutation

  • It includes the following that can affect base pairing in a gene: a. Deletion b. Insertion c. Inversion d. Translocation
  • Even a single base pair can affect mutation
  1. Point mutation
  • Alterations of a single or a few base pairs.
  • Point mutations are increasingly analyzed by sequencing methods.
  • Sequencing not only direct detects the mutated base or bases but also provides the context of neighboring bases.
  • Detected by NGS Method over large sequence regions have to be screened to distinguish among silent, conservative and non-conservative changes.
  • Phenotypic alterations in protein structure can only be predicted from the nucleotide sequence.
  • May or May not change the encoded amino acid. ● Point mutation on 3’ End
    • Found in ending of coding sequence.
    • Causes minimal consequences. ● Proof-reading
      • DNA polymerase has the ability to proof read sequence.
      • Removes mutated or non-complementary bases. ● Point mutation on 5’ End
    • Found in beginning of coding sequences
    • Alterations or deletion.

Types of Gene Mutation

  • About 11% of diseases-related gene lesions are nonsense mutations. ● Non-conservative, Nonsense and Frame shift mutations
    • Will generate different phenotypes, depending on where they occur along the protein sequence.
  • Amino acid codon ฀ important concept for interpreting the results of mutation analyses.
  1. Silent Gene Mutation
  • Substitution of one nucleotide with a different nucleotide.
  • Eg. GCC (alanine)
    • Mutation to GCA
  • Changes in DNA sequence BUT it does not change the amino acid sequence.
  1. Conservative Gene Mutation
  • May change the amino acid sequence, but the replacement and the original amino acid have similar biochemical properties.
  • Eg. GTG (Leucine) ฀ GUG (Valine)
  1. Non-Conservative Gene Mutation
  • Replacement of an amino acid with biochemically different biochemical properties of amino acid.
  • Eg. Proline ฀ Glutamine
  1. Non-Sense Gene Mutation
  • Terminates proteins prematurely when a nucleotide substitution produces a stop codon instead of an amino acid codon.
  • Changes in phenotypic of organism.
  • 3 Stop codons: a. UAG b. UGA c. UAA
  1. Frameshift Gene Mutation
  • Insertion or deletion of other than multiple of three nucleotides.

Detection of Gene Mutations

  • Detection of mutations in large genes requires screening across thousands of base pairs to detect a single altered nucleotide.
  • Advances in sequencing technology allow genome-wide scanning for yet-unreported mutation.
  • Eg. Factor 5 laiden mutation and Hemochromatosis
    • Have the same genetic location.

I. Biochemical Methods (Nucleic Acid Analysis)

  • Analysis: Checks for the protein product.
  • Used to directly analyze the change in protein structure or function rather than to search for potential point mutations.
  • Detects metabolic defects and protein or amino acid alterations.
  • Checks the phenotypic characteristic or the encoded protein.
  • Use to detect the altered protein product or phenotype. ● Phenotypic characteristic
    • It is based on the genome of organism.
  • Commonly used biochemical methods:
    1. Immunoassays
    • For detection of hormone, drugs, antibodies, cancer biomarkers, and metabolites.
    1. Immunohistochemistry
    • Detects protein abnormalities in situ or inside the cell.
    • Pathologist are the one who reads the result.
    1. High-Performance liquid or Gas Chromatograpahy (HPLC/GC)
    • Automated methods.
  1. Mass Spectrometry
  2. Enzyme Immunoassays
  • Enzyme immunoassay formats for direct antigen detection.
  • Involve the use specific antibodies or other ligands to detect the presence of the target molecules.
  • Utilize or uses the following with coated with capture antibody: a. Plate wells b. Strips c. Capillaries
  • If the analye is present the test fluid, It will bind to the immoboiized antibody.
  • Dectections by the use of secondary antibody conjugated to enzyme.
  • Eg. Alkaline Phosphatase (AP) / Horse raddish peroxidase (HPO) ฀ Chemiluminesence, fluoresence or color signals. ● Secondary antibody
  • It is conjugated to Alkaline phosphatase or Horse raddish peroxidase.
  • Forms a sandwhich formation.
  • Specific to antigen-antibody complex. ● Antigen detection
  • Uses immobilized antibody conjugated with enzyme.
  • Uses samples like urine, plasma and serum.
  • Eg. AP and HPO ● Antibody detection
  • Uses immobilized antigen and antihuman antibody and antihuman antibody conjugates. ● Substrate
  • Produces color generation.
  1. Immunohistochemistry
  • Performed on thin (<5 micron) slices of fixed or 5-15 microns slices of frozen tissue.
  • It provides the advantage of integrating target detection, localization and quantification in the context of tissue morphology.
  • Uses Imaging or microscopic observation of antibody binding ฀ Fluorescent or colorimetric
  • 2 Secondary antibody: a. Fluorescein (Fluorescent)
  • Use to check fluorescent signal.
  • Bound to antibodies when the floor is excited. b. AP or HRP (Colorimetry)

II. Nucleic Acid Analyses

  • Analysis: Location of gene mutation in a sequence.
  • Mutation detection by analysis of nucleic acids is considered the classical molecular methodology.

Types of mutation

  1. Somatic Mutation
  • Occur in a single body cell and cannot be inherited.
  • Only tissue derived from mutated cells are affected.
  1. Germline Mutation
  • Occur in gametes and can be passed onto offspring.
  • All cells have mutations.
  • Non-invasive or convenient. ● Buccal epithelial cells or Blood
    • Ideal source of sample.

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

  1. Hybridization-based methods a. Single-strand conformation of polymorphism (SSCP) b. Allele-Specific Oligomer Hybridization c. Melt- Curve Analysis (MCA) d. High Resolution Melt-Curve Analysis (HR-MCA) e. Heteroduplex Analysis f. Array Technology

  2. Sequence (polymerization) based methods a. Sequence-specific (Primer) PCR (SS- PCR) b. Allelic Discrimination with Fluorogenic Probes

  3. Enzymatic or Chemical cleavage methods a. Restriction Fragment Length Polymorphism (RFLP) b. Non-isotropic RNase cleavage assay (NIRCA) c. Cleavase Assay

  4. Hybridization-based methods

  5. Single-Strand Conformation of Polymorphism (SSCP)

  • Is based on the preference of DNA (as well as RNA) to exist in a double stranded.

  • SSCP is reported to detect 35% to 100% of putative. ssDNA ฀ No complementary strand ฀ Intrastrand Duplexes (hair pin formation) ● Intrastrand Duplexes

    • Hair pin formation.
    • Produces duplexes on it own. ● Conformer
    • A folded strand (3D structure)
    • The shape of which is determined by the primary sequence of the folded strand.
    • Migratio single-stranded conformers in polyacrylamide gels/capillary electrophoresis ฀ Distinguises sequences variants.

  • Heavier: Normal sequence/ More folds

  • Lighter: Mutated sequence/ Less folds

SSCP Procedure

  1. Denaturation of short, dsPCR products (100-400 bases) a. 10 to 20 mM NaOH b. 80% Formamide or a. 10 to 20 mM NaOH b. 0 mM EDTA c. 10% Formamide
  2. Rapid cooling
  3. Diluted single strands cannot easily find their homologous partners, They fold intrastrand hybridization ฀ Forming 3D conformers
  • A single-base difference in the DNA sequence can cause the conformers to fold differently.
  1. Conformers are resolved in a polyacrylamide gel or by capillary electrophoresis with strict temperature control.
  • The speed of migration depends on the shape as well as the size of the conformer.
  • ↑ Folds: Slow migration
  1. The band or peak patterns are detected by: a. Silver stain b. Radioactivity c. Fluorescence

Steps SSCP Procedure

  1. Denature.
  2. Rapid cooling
  3. Formation of conformer
  4. Polyacrylamide gel electrophoresis

  • Normal: Heavier

  • Mutant: Lighter

  • Mutant: Consist of 4 bands

  1. Capillary electrophoresis

  2. Allele-Specific Oligomer Hybridization

  • Utilizes the differences in the melting temperatures of short sequences with one or two mismatches and those with no mismatches.
  • Synthetic single-stranded probes with the normal or mutant target DNA sequence are used for this assay. ● Probe
    • WILL NOT BIND to near complementary target sequence with one or two mismatched bases.
    • Perfect complementary sequence will bind.
  • Methods used: a. Dot Blot Method
    • Uses an immobilized target and a labelled probe in solution. b. Reverse Dot Blot Method
    • Uses 96 well plate method.
    • Mutant or normal probes were immobilized on a membrane.
    • Denatured biotinylated amplicons ฀ Immobilized probes
    • Only the exact complementary

sequences would hybridize. - Bound probes ฀ Detected using HPO-Antibiotin Fab fragment.

  • Generation of a color light signal indicated the blinding of the test DNA to the normal or mutant.
  1. Melt- Curve Analysis (MCA)
  • Post-amplification step of aPCR.
  • Exploits the sequence and stacking-directed denaturation characteristics of DNA duplexes.
  • PCR amplicons generated in the presence of a DNA-specific fluorescent dye: a. Ethidium bromide b. SYBR green
  1. Array Technology
  • Analysis: Fragments
  • Single-base-pair resolution by hybridization differences is achievable with high-density oligonucleotide arrays.
  • The test DNA is fragmented by treathment with DNase before binding to the complementary probes on the arrays.
  • After hybridization of the fluorescently labeled sample DNA, ฀ Fluorescent signal (software scanner) ฀ Mutations are identified as indicate which probes are bound.

2 Types of Hybridization format

  • Binding of the sample fragment is detected by INCREASE fluorescence.
  1. Standard tiling
  • The base substitution in the immobilized probe is always in the 12th position from its 3’ end.
  1. Redundant tiling
  • Same mutation is placed at different positions in the probe (at the 5’ end, in the middle, or at the 3’ end.)
  1. Sequence (polymerization) based methods
  2. Sequence-specific (primer) PCR (SSP-PCR)
  • Analysis: Presence or Absence or product.
  • Involves careful design of primer.
  • Commonly used to detect point mutations and other SNPs.
  • 3’ end of a primer must match the template perfectly to be extended by Taq polymerase.
  • The presence or absence of product is interpreted as the presence or absence of the mutation.
  • Presence of product: Mutation

Modification of SSP-PCR

  1. Allele-specific primer amplification
  • Increasing the length of the normal or the mutant primer, resulting in differently sized products.
  1. Multiplex allele-specific PCR
  • Using multiple primers to detect multiple PCR products.
  • Originally called Amplification Refractory Mutation System PCR or Tetraprimer PCR.
  1. Allelic Discrimination with Fluorogenic Probes
  • Real-time PCR assay using two probes. ฀ 3’ Quencher ฀ 5’ Flour
  • Each probe is complementary to either the normal or mutant sequence.
  • The hybridized probe is digested by the polymerase enzyme.

Steps in Allelic Discrimination with Fluorogenic Probes

  1. The probe complementary to the normal sequence is labeled with FAM dye.
  2. The probe complementary to the mutant sequence is labedled with VIC dye.

  • If the test sequence is NORMAL ฀ FAM fluorescence will be high and VIC fluorescence will be low. (↑ FAM fluorescence: ↓ VIC fluorescence)

  • If the test sequence is MUTANT (↓ FAM fluorescence: ↑ VIC fluorescence)

  • If the sequence is heterozygous both FAM dye and Vic Dye will be HIGH.

  1. Enzymatic and Chemical Cleavage Method
  2. Restriction Fragment Length Polymorphism (RFLP)
  • Mutation changes the structure of a restriction enzyme target sites or changes in the size of fragment generated by a restriction ezyme.
  • RFLP analysis can be used to detect the sequence alteration.
  • To perform PCR-RFLP, the region sorrounding the mutation is amplified, and the mutation is

detected by cutting the amplicon with the appropriate restriction enzyme.

  • Mutations may inactive a naturally occuring restriction site or generate a new restriction site so that digestion of the PCR product.
  • Resulting in cutting of the mutant amplicon, but not a normal control amplicon or vice versa.
  1. Non-isotropic RNase cleavage assay (NIRCA)
  • Heteroduplex analysis using duplex RNA.
  • The sequences to be scanned are amplified using primers tailed with promoter sequences of 20 to 25 bp.
  • The following amplification ฀ PCR products with promoter sequence are used as templates for in vitro synthesis of RNA.
  • Using T7 or SP6 RNA polymerase enzymes. ฀ This reaction yields a large amount of double-stranded RNA.
  1. Cleavase Assay

  • Based on the characteristic enzymatic activity of a proprietary enzyme system (cleavase)

  • Pre-mixed reagents with cleavase + Sample (DNA/ PCR product/ Amplicon) + Control

  • The Human Genome Organization (HUGO) gene nomeclature committee has set rules for reporting or publishing gene names.

  • Gene names should be CAPITALIZED and set in in Italics with NO hyphens.

  • Protein names are NOT ITALICIZED nor COMPLETELY CAPITALIZED -Example: a. KRAS gene cods for the K-Ras protein b. TP53 gene codes for the protein p

Was this document helpful?

Gene Mutation and Mutation Detection Technique

Course: College of Medicine

291 Documents
Students shared 291 documents in this course

University: La Consolacion University Philippines

Was this document helpful?
Gene Mutation and Mutation
Detection Technique
- AKA “Gene Variant”.
- A permanent change in a DNA sequence.
- Mutations arise spontaneously at low
frequency owing to the chemical instability of
the purine and pyramidine bases.
1. Mutagenic
- Overexpose to ultraviolet light.
2. Chemical carcinogens
- Eg. Aflatoxin B1 can cause mutation.
Gene Mutation
- It includes the following that can affect base
pairing in a gene:
a. Deletion
b. Insertion
c. Inversion
d. Translocation
- Even a single base pair can affect mutation
1. Point mutation
- Alterations of a single or a few base pairs.
- Point mutations are increasingly analyzed by
sequencing methods.
- Sequencing not only direct detects the mutated
base or bases but also provides the context of
neighboring bases.
- Detected by NGS Method over large sequence
regions have to be screened to distinguish
among silent, conservative and non-conservative
changes.
- Phenotypic alterations in protein structure can
only be predicted from the nucleotide sequence.
- May or May not change the encoded amino
acid.
●Point mutation on 3’ End
- Found in ending of coding sequence.
- Causes minimal consequences.
●Proof-reading
- DNA polymerase has the ability to
proof read sequence.
- Removes mutated or
non-complementary bases.
●Point mutation on 5’ End
- Found in beginning of coding sequences
- Alterations or deletion.
Types of Gene Mutation
- About 11% of diseases-related gene lesions are
nonsense mutations.
●Non-conservative, Nonsense and Frame
shift mutations
- Will generate different phenotypes,
depending on where they occur along the
protein sequence.
- Amino acid codon ฀important concept for
interpreting the results of mutation analyses.
1. Silent Gene Mutation
- Substitution of one nucleotide with a different
nucleotide.
- Eg. GCC (alanine)
- Mutation to GCA
- Changes in DNA sequence BUT it does not
change the amino acid sequence.
2. Conservative Gene Mutation
- May change the amino acid sequence, but the
replacement and the original amino acid have
similar biochemical properties.
- Eg. GTG (Leucine) ฀GUG (Valine)
3. Non-Conservative Gene Mutation
- Replacement of an amino acid with
biochemically different biochemical properties
of amino acid.
- Eg. Proline ฀Glutamine
4. Non-Sense Gene Mutation
- Terminates proteins prematurely when a
nucleotide substitution produces a stop codon
instead of an amino acid codon.
- Changes in phenotypic of organism.
- 3 Stop codons:
a. UAG
b. UGA
c. UAA
5. Frameshift Gene Mutation
- Insertion or deletion of other than multiple of
three nucleotides.

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