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 16^th 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