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Sunday, January 06, 2008

detect the point mutation in the beta (b) – globin gene that causes sickle cell anemia

006. The following methods can be used to detect the point mutation in the beta (b) – globin gene that causes sickle cell anemia, except:

1. Polymerase chain reaction with allele-specific oligonucleotide hybridization.

2. Southem blot analysis.

3. DNA sequencing.

4. Northern blot analysis.

Answer

4. Northern blot analysis.

Reference:

Harrison 16th Edition Page 378

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Discussion

Methods Used for the Detection of Mutations

Method

Principle

Type of Mutation Detected

Cytogenic analysis

Unique visual appearance of various chromosomes

Numerical or structural abnormalities in chromosomes

Fluorescent in situ hybridization (FISH)

Hybridization to chromosomes with fluorescently labeled probes

Numerical or structural abnormalities in chromosomes

Southern blot

Hybridization with genomic probe or cDNA probe after digestion of high molecular DNA

Large deletion, insertion, rearrangement, expansions of triplet repeat, amplification

Polymerase chain reaction (PCR)

Amplification of DNA segment

Expansion of triplet repeats, variable number of tandem repeats (VNTR), gene rearrangements, translocations; prepare DNA for other mutation methods

Reverse transcriptase PCR (RT-PCR)

Reverse transcription, amplification of DNA segment absence or reduction of mRNA transcription

Analyzed expressed mRNA (cDNA) sequence; detect loss of expression

DNA sequencing

Direct sequencing of PCR products

Sequencing of DNA segments cloned into plasmid vectors

Point mutations, small deletions and insertions

Single-strand conformational polymorphism (SSCP)

PCR of DNA segment: Mutations result in conformational change and altered mobility

Point mutations, small deletions and insertions

Denaturing gradient gel electrophoresis (DGGE)

PCR of DNA segment: Mutations result in conformational change and altered mobility

Point mutations, small deletions and insertions

RNAse cleavage

Cleavage of mismatch between mutated and wild-type sequence

Point mutations, small deletions and insertions

Restriction fragment length polymorphism (RFLP)

Detection of altered restriction pattern of genomic DNA (Southern blot) or PCR products

Point mutations, small deletions and insertions

Oligonucleotide specific hybridization (OSH)

Hybridization of PCR products to wild-type or mutated oligonucleotides immobilized on chips or slides

Point mutations, small deletions and insertions

Microarrays

Hybridization of PCR products to wild-type or mutated oligonucleotides

Point mutations, small deletions and insertions

Protein truncation test (PTT)

Transcription/translation of cDNA isolated from tissue sample

Mutations leading to premature truncations

Explanation

Northern blot is a technique used for analysis of RNA

Comments

Western blot is used for the analysis of proteins

Tips

Southern blot was named after the person who devised the technique, and the other names began as laboratory jargon but are now accepted terms

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