DNA and Chromosomal Analysis

Question 1

What are the 3 methods of testing for point mutations?

Answer 1

Automated fluorescent dideoxy (Sanger) DNA sequencing

Massively parallel/next generation DNA sequencing

Allele-specific (ARMS) PCR

Question 2

Briefly outline Sanger DNA sequencing.

Answer 2

Tests 1 gene at a time

Shows all bases on the gene

Question 3

What is the function of next generation DNA sequencing?

Answer 3

Tests many genes at once

Question 4

What is the function of allele-specific (ARMS) PCR?

Briefly describe the method.

Answer 4

To test for specific point mutations - i.e. you need to know what you’re looking for

PCR primers are added which only bind to the specific mutation if present
Therefore DNA will only replicated if the mutation is there

Question 5

Briefly describe the process of PCR.

Answer 5

1. Starts with 1 copy of DNA, with a sequence of interest surrounded by 2 known sequences
2. Primers bind to known sequences, begin replicating sequence of interest
3. Each round doubles number of DNA copies; usually 30 rounds are done

Question 6

What are the 2 methods of detecting sub-microscopic duplications or deletions of DNA?

Answer 6

MLPA (PCR-based)

Array comparative genomic hybridisation (aCGH)

Question 7

What does MLPA test for?

Answer 7

Deletion of one or more genes (less than 4 million base pairs)

NOTE: need to know which mutation you’re looking for

Question 8

What does aCGH test for?

Answer 8

Duplications or deletions across the whole genome

Question 9

Briefly outline the process of an aCGH test.

Answer 9

1. Compares the patient’s DNA to a set of reference DNA
2. This shows which set has more DNA present:
   a. If patient has more DNA, there is a duplication mutation
   b. If reference has more DNA, there is a deletion mutation

Question 10

What are the 3 methods of quickly testing for aneuploidies?

Answer 10

Quantitative fluorescent PCR (QF-PCR)

Karyotyping

Fluorescent in situ hybridisation (FISH)

Question 11

What will QF-PCR show you?

Answer 11

Comparison of different chromosome copies to detect trisomies

Normally there should be two peaks; if there are 3 there is a trisomy

Question 12

What will FISH show you?

Answer 12

How many copies there are of a particular gene

i.e. need to know which gene you’re looking for, and its exact location

A probe is added to bind to a particular gene; how many times it binds shows how many copies are present

Question 13

Briefly outline the illumina method of next generation sequencing.

Answer 13

1. Machine takes a picture of each nucleotide, showing in a different colour, thus showing the sequence of bases in a DNA sequence
2. Machine produces FASTQ file of raw data containing reads (strings of base letters)
3. Machine aligns reads onto a reference genome to create a BAM file
4. Computer shows which parts of patient’s DNA are different to the reference; creates a variant cell format (VCF) file

Question 14

What is the main ethical issue with next generation DNA sequencing?

Answer 14

Incidental findings - should the patient be told?

e.g. if testing for a Huntington’s mutation, should the patient be told about a BRCA2 mutation if found?