5: Mutation and Detection Flashcards by Ma. Clariza Luna

Mutation or Polymorphism

A single gene locus causing mutation has a major physiologic impact and considered to be deterministic of a disease

Mutation

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Mutation or Polymorphism

Seen less commonly in general population.

Mutation

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Mutation or Polymorphism

Linked to Mendelian disease.

Mutation

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Mutation or Polymorphism

Correlation on between mutation and disease can be established.

Mutation

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Mutation or Polymorphism

Single base pair change in nucleotide sequence of genes are called point mutation.

Mutation

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Mutation or Polymorphism

Genetic alteration that contributes to complex disease has smaller effect.

Polymorphism

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Mutation or Polymorphism

Present in at least 1% of the population

Polymorphism

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Mutation or Polymorphism

No correlation between mutation and disease

Polymorphism

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Mutation or Polymorphism

Single base pair change in nucleotide sequence of genes

Polymorphism

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In mutation, single base pair change is called?

point mutation.

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In polymorphism, single base pair change is called?

single nucleotide polymorphism (SNP)

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What are the types of base pair mutation?

Transition
Transversion
Deletion
Insertion

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Type of base pair mutation:

T-A to C-G; pyrimidine to pyrimidine or purine to purine

Transition

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Type of base pair mutation:

CATCCACCTGTACCA
GTAGGTGGACATGGT

[normal sequence:
CATTCACCTGTACCA
GTAAGTGGACATGGT]

Transition

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Type of base pair mutation:

T – A to G – C; pyrimidine to purine

[normal sequence: CATTCACCTGTACCA GTAAGTGGACATGGT]

Transversion

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Type of base pair mutation:

can involve more than one base pair

CATGACCTGTGCCA
GTACTGGACACGGT

[normal sequence:
CATTCACCTGTACCA
GTAAGTGGACATGGT]

Deletion

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Type of base pair mutation:

can involve more than one base pair

CATCACCTGTACCA
GTAGTGGACATGGT

[normal sequence:
CATTCACCTGTACCA
GTAAGTGGACATGGT]

Insertion

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What are the two types of mutation?

Spontaneous mutation
Induced mutation

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Type of mutation:

Internal and cannot be controlled

Spontaneous mutation

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What are the types of SPONTANEOUS MUTATION?

Hydrolytic reaction, Replication error

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types of SPONTANEOUS MUTATION:

Hydrolysis occurs within part of base pair

Hydrolytic reaction

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types of SPONTANEOUS MUTATION:

tautomer formation

Replication error

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Replication error:

C-G→C-G and C-G

normal replication of C-G should result to two daughter strands of C-G

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Replication error:

C-G→C-A and C-G

tautomer formation results in mispairing and insertion of A

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Type of mutation: external and controllable

Induced Mutation

What are the 3 examples of INDUCED MUTATION?

1. UVL 2. Ionizing radiation 3. Chemical mutagen

Example of Induced mutation: Formation of thymine dimer by UV light

UVL

Example of Induced mutation: X rays, ɣ rays (gamma rays), and β particles which can significantly damage our DNA.

Ionizing radiation

Example of Induced mutation: deamination of nitrous acid

Chemical mutagen

What happens when chemical mutagen occurs in Cytosine?

1. Cytosine is deaminated to uracil 2. Uracil binds with Adenine

What happens when chemical mutagen occurs in Adenine?

1. Adenine is deaminated to Hypoxanthine 2. Cytosine binds with Hypoxanthine

→ Also known as SNP. → read as “snip” → a change in a single nucleotide in the genome that causes variations in DNA sequences between members of the same species. → occurs when two individuals in the population differ by a single base in the DNA sequence. → lighter burden

Single Nucleotide Polymorphism

Factors to consider in polymorphism?

→ Present in more than 1% of the population → other wise, Variation is a rare mutation

T or F Single Nucleotide Polymorphism is not normal and when you have it you should get checked

F (its normal bb)

T or F Single Nucleotide Polymorphism occurs normally throughout a person’s DNA (3 X 10^9 bp).

T or F Single Nucleotide Polymorphism commonly found in the DNA between genes.

How many Single Nucleotide Polymorphism is in a person's entire genome?

4 - 5 M SNPs

T or F SNP is present almost once in every 5,000 nucleotides

F (almost once in every 1,000 NUCLEOTIDES)

T or F SNP can be caused by induced mutation over time

F (caused by SPONTANEOUS MUTATION)

Importance of SNP?

→ important in studying human health → may predict individuals’ response to drugs, susceptibility to environmental factor, risk to a particular disease, association with complex disease → track inheritance of disease (pedigree)

What does not belong? a. SNP occurs normally throughout a person’s DNA (3 X 10^9 bp) b. caused by spontaneous mutation over time c. track inheritance of disease d. ALL of the aboce e. NOTA

c. track inheritance of disease (c is importance of SNP; other choices answers the question is SNP normal)

Single Nucleotide Polymorphism Facts?

→ act as biological markers → occurs within a gene that is described as having more than one allele. → May lead to variations in amino acid sequence

3 types of genetic disorders

1. Single Gene Disorder 2. Multifactorial Disorders 3. Chromosomal Disorder

Genetic Disorder: caused by a single gene, several different mutations can result in the same disease but with varying degrees of severity and phenotype.

Single Gene Disorder

Genetic Disorder: main factor is genes, but the cause includes other factors that aren’t genes.

Multifactorial Disorders

Genetic Disorder: abnormal chromosome number or constitution

Chromosomal Disorder

Mutation Detection Techniques? Chromosomal

1. Conventional Karyotyping 2. Fluorescent in situ Hybridization (FISH) 3. Spectral Karyotyping (SKY)

Identify what mutation detection technique: → chromosome abnormality → indicative in: sexual disorders, congenital anomalies, developmental retardation, learning disabilities, infertility, multiple miscarriage, stillbirth, malignancy,

Conventional Karyotyping

Identify what mutation detection technique: → provide specific localization of genes on chromosomes → indicative in: trisomy and microdeletion using specific probes

Fluorescent in situ Hybridization (FISH)

Identify what mutation detection technique: → homologous pairs of chromosomes are manipulated in such a way that they have distinctive colors.

Spectral Karyotyping (SKY)

Mutation Detection Techniques under molecular diagnostics?

1. Hybridization Based 2. Sequencing (Polymerization) 3. Cleavage Based

Under hybridization based molecular diagnostics, what are the methods of molecular diagnostic?

1. Fluorescent in situ Hybridization (FISH) 2. Allele-specific Oligonucleotide (ASO) 3. Array-based Comparative Genomic Hybridization (aCGH) 4. Array Technology

Hybridization based molecular diagnostics method: → hybridization between the ssDNA and target gene with probe. → Can be applied in both chromosomal studies and molecular diagnostics.

Fluorescent in situ Hybridization (FISH)

Hybridization based molecular diagnostics method: → "dot blot method" → relies on binding effects of nucleotide mismatches → stringent conditions are applied so that a single- base mismatch is sufficient to prevent hybridization of the non-matching probe

Allele-specific Oligonucleotide (ASO)

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) are applied so that a single- base mismatch is sufficient to prevent hybridization of the non-matching probe

Stringent conditions

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) How many probes does ASO require?

2 probes, one specific for each allele

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) + probes is complimentary to what?

normal sequence

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) m probes is complimentary to what?

mutant sequence

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) What is the result for individual 1?

Normal

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) What is the result for individual 2? [insert image]

Normal individual; since +/+ tapos may + probe lang may dot

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) What is the result for individual 3? [insert image]

Presence of mutation; since sa may m probe lang may dot

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) What is the result for individual 2 [insert image 2]

Heterozygous individual; since +/-

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) What is the result for individual 2? [insert image 2]

Heterozygous since tapos may dot sa both

Hybridization based molecular diagnostics: Allele-specific Oligonucleotide (ASO) what does m, +, N mean at the end of the photo? [insert image 2]

controls m - mutant control + - normal control N - negative control

Hybridization based molecular diagnostics method: → a special FISH technique (dual probes) → detecting all genomic imbalances → comparison of total genomic DNA of given sample with total genomic DNA of normal cells

Array-based Comparative Genomic Hybridization (aCGH)

Hybridization based molecular diagnostics: Array-based Comparative Genomic Hybridization (aCGH) What are the steps?

1. Identical amount of both tumor and normal DNA is labeled with two different fluorescent dyes and mixed (Put fluorophone) 3. Mixture is added and hybridized to a normal lymphocyte metaphase slide. 4. A fluorescent microscope is used for evaluation

Hybridization based molecular diagnostics: Array-based Comparative Genomic Hybridization (aCGH) How many fluorescent dyes are used?

2, 1 for each sample

Hybridization based molecular diagnostics method: Used to determine copy number alterations (if there are amplification or deletion) of genome in cancer and those cells whose karyotype is hard or impossible to prepare or analyze.

Array-based Comparative Genomic Hybridization (aCGH)

Hybridization based molecular diagnostics: Array-based Comparative Genomic Hybridization (aCGH) What color in the result means Tumor & Normal

Yellow

Hybridization based molecular diagnostics: Array-based Comparative Genomic Hybridization (aCGH) What color in the result means Tumor & Tumor

Red

Hybridization based molecular diagnostics: Array-based Comparative Genomic Hybridization (aCGH) What color in the result means Normal & Normal

Green

Hybridization based molecular diagnostics method: → reverse dot blot method (based on matching) → investigate multiple genomic sites. → unlabeled probes are bound to substrate → specimen DNA is labeled and hybridized to immobilized probes.

Array Technology

Which does not belong in Array Technology a. Detection b. Method c. Substrate d. Enzyme e. ALL of the above f. NOTA

d. Enzyme

How many methods are given in Array Technology

Hybridization based molecular diagnostics: Array Technology What are the methods under array tech?

1. Microarray 2. Macroarray 3. High Density oligonucleotide arrays 4. Microelectric arrays 5. Comparative genomic hybridization 6. Expression array 7. SNP detection, mutation analysis, sequencing

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of Macroarray method?

Substrate: Nitrocellulose Detection: Radioactive, Chemiluminescent, Chromogenic

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of Microarray method?

Substrate: Glass, Nitrocellulose on glass Detection: Fluorescent

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of High Density Oligonucleotide array method?

Substrate: Glass Detection: Fluorescent

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of Microelectronic arrays

Substrate: Electro grid Detection: Fluorescent

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of Comparative Genomic Hybridization

Substrate: Micro and Macro array Detection: Detection of genomic amplifications and deletions

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of Expression array

Substrate: Micro and Macro array Detection: Detection of relative changes in gene expression

Hybridization based molecular diagnostics: Array Technology Substrate and Detection of SNP Detection, analysis, sequencing

Substrate: High Density Oligonucleotide Array Detection: Detection of single- base differences in DNA

Sequencing (polymerization) comprise of what methods

1. DNA sequencing 2. Allele specific PCR

Sequencing (polymerization) method: → allows rapid detection of SNP or mutation. → less laborious → uses a chromatogram

DNA Sequencing

Sequencing (polymerization) how many SNP are detected?

1 specific SNP

Sequencing (polymerization) method: DNA Sequencing What results are shown?

spikes as result

Sequencing (polymerization) method: → application of PCR that allows the direct detection of any point mutation, single base changes or small deletions in human DNA → involves amplification of DNA with single nucleotide polymorphism (SNP) → uses SNP-specific primers → under stringent conditions PCR is less efficient in the presence of mismatch between the template and primer → Successful amplification with an SNP-specific primer signals the presence of the specific SNP in a sequence

Allele specific PCR

Sequencing (polymerization) method: Allele specific PCR T or F The Allele specific PCR uses DNA probes

F (SNP-Specific primers)

Sequencing (polymerization) method: Allele specific PCR If a normal primer is used to normal allele, what is the result?

Amplification

Sequencing (polymerization) method: Allele specific PCR If a mutant primer is used to mutant allele, what is the result?

Amplification

Sequencing (polymerization) method: Allele specific PCR If a mutant primer is used to normal allele, what is the result?

No amplification

Sequencing (polymerization) method: Allele specific PCR If a normal primer is used to mutant allele, what is the result?

No amplification

Sequencing (polymerization) method: Allele specific PCR T or F The primers are non-specific to the alleles in Allele specific PCR

F (primers are SPECIFIC)

Cleavage based type of Mutation Detection Technique comprise of what method?

Restriction Fragment Length Polymorphism (RFLP)

Cleavage-based: → a technique that is used to study genetic variation or polymorphisms among individuals using restriction enzymes. → any genomic DNA can be differentiated according to the presence or absence of restriction enzyme sites.

Restriction Fragment Length Polymorphism (RFLP)

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) T or F Restriction enzymes recognize and cut at a random site.

F (recognize and cut at PARTICULAR SITE)

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) Steps?

1. Restriction Digest 2. Electrophoresis 3. Denaturation 4. Blotting 5. Baking and Blocking 6. Probe hybridization and visualization

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) What gel is used in electrophoresis?

Agarose or Polyacrylamide

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) What reagent is used in denaturation?

NaOH

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) What blotting and membrane is used in blotting?

Souther blotting, Nitrocellulose paper

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) What procedures are done for baking and blocking?

Autoclave, Bovine Serum Albumin or Casein

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) This is put in baking and blocking step in order to fix DNA

Autoclaving

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) process that prevents binding of labelled probe non-specifically to charged membrane

Blocking

Cleavage-based: Restriction Fragment Length Polymorphism (RFLP) What is used in probe hybridization and Visualization

labelled RFLP probe, autoradiography

Application of Restrction Fragment Length Polymorphism?

1. Crime scene 2. Paternity 3. Diseases