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Biochemistry

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Purines, Pyrimidines and Nucleic Acid Metabolism

Question
25 out of 59
 

Chromosomal mutations can be identified from all except (AIIMS Nov 2010)



A Single stranded polymorphism

B Dideoxy nucleotide trail sequencing

C Agarose gel electrophoresis

D Denaturing Gradient Gel Electrophoresis (DGGE)

Ans. C Agarose gel electrophoresis

a. Denaturing Gradient Gel Electrophoresis (DGGE):

b. DNA is first extracted either from paraffin embedded or fresh tissues and subjected to denaturing gradient gel electrophoresis. The method detects selective retention of mutated BRCA1 alleles, based on the principle that different sequences of double-stranded DNA melt under different conditions.

c. A modified form of DGGE is called as TGGE (Temperature Gradient Gel Electrophoresis) which uses temperature as a denaturing agent

Nucleotide sequencing:

In this method, the complete gene is sequenced to identify the presence of any mutation in breast cancer. Till date complete gene sequencing is considered as a gold standard method because any type of mutation can be identified. Sequencing is done by two methods:

  1. Sanger's dideoxy nucleotide method
  2. Shotgun method

Sanger Gene Sequencing: This method looks for point mutations (a change from one base pair to another), or deletions or insertions (of several bases, as opposed to an entire exon) in the dystrophin gene. It can miss a few rare mutations. Sequencing can be used to locate small mutations in female carriers.

Resequencing Array: This test is a chip-based sequencing method. It looks for point mutations (a change from one base pair to another), or deletions or insertions (of several bases, as opposed to an entire exon) in the dystrophin gene. Array-based sequencing may include the entire coding region of the dystrophin gene, plus the gene promoters and some reported intronic mutations. Females can be carrier tested for small mutations using this method.

Single Strand Conformational Polymorphism:

a. This method can detect single base pair mutations like frameshift mutations, nonsense mutations and missense mutations in breast cancer. The basic principle of this test is that double stranded DNA when denatured, assumes a special conformation.

b. This conformation is unique and depends on primary nucleotide sequence. This method is quite sensitive to detect even single nucleotide difference that occupies a different conformation and when subjected to electrophoresis, the variant nucleotide occupies a different position.

Purines, Pyrimidines and Nucleic Acid Metabolism Flashcard List

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