Chromosome, DNA, and clinical genomic analysis

Question 1

Describe Sanger sequencing

Answer 1

Sequences one gene at a time.

Chain-terminating nucleotides which have been tagged with a different colour dye are added into the normal PCR method (e.g. normal nucleotides)

Nucleotides are added onto the primer until the template is completed.

Fragments are run through gel electrophoresis (short sequences move quickly, big move slowly). A laser can then illuminate the sequence.

Question 2

Describe next generation sequencing.

Answer 2

Sequences many genes at once, and used in detection of point mutations.

Similar to Sanger, but on bigger scale as many genes are sequences at one.

Example is Illumina method.

Question 3

Describe allele-specific PCR: ARMS.

Answer 3

Used in detection of specific, known point mutations.

Primers called ASOs which are specific at the 3’ end for a mutant sequence.

PCR amplification occurs once the 3’ end has paired with the template DNA.

Question 4

Describe multiplex ligation-dependent probe amplification (MLPA)

Answer 4

Used in detecting duplications and deletions affecting specific genes.

PCR is used to analyse the copy number along the DNA sequence.

Used in conditions with deletions/duplications affecting >1 exon e.g. DMD, BRCA1/2, and when mutations are too small to be detected by FISH.

Question 5

Describe array comparative genomic hybridisation (aCGH)

Answer 5

Used in detecting duplications and deletions.

Compare DNA to a reference at different points in the genome.

DNA is colour labelled and compared to microarrays of specific DNA sequences, and the unbound DNA is washed away.

Question 6

Describe QFPCR

Answer 6

Used to detect aneuploidy in chromosomes 13, 18, and 21.

Tetra nucleotide repeats are amplified using PCR using fluorescent primers, and then amplified.

Trisomies are detected by the presence of 3 peaks instead of 2 e.g. 3 alleles of a gene are present.