methods of molecular biology 2

Question 1

how do we visualise small scale DNA mutations?

Answer 1

with PCR, sequencing, array technologies

Question 2

how do we visualise large scale DNA mutations?

Answer 2

G-banding, FISH, and array CGH

Question 3

how do we perform karyotyping?

Answer 3

1. cells stimulated to divide in culture
2. dividing cells arrested in metaphase - cells burst open using hyptonic solution
3. fixation and dropping of cells onto microscope slide
4. Giemsa staining (G-banding)
5. examine/photagrpah via microscope

produces light (euchromatin) and dark bands (heterochromatin)

Question 4

what is FISH?

Answer 4

flourescent In-situ hybridisation (FISH)

it is a method to visualize specific regions of DNA in an individual cell - including genes or portions of genes

it has greater resolutions/is more sensitivie than G-banded karyotyping

it paints chromosomes or portions of chromosomes with fluorescent molecules- can determine the presence and location of a region of DNA within chromosome preparations, fixed cells or tissues - can detect duplications or deletions of DNA smaller than detected by G-banded karyotype

Question 5

what are the limitations of FISH?

Answer 5

1. it is specific to what you design the probes for - FISH can only detect deletions or duplications of regions - specifically targeted by the probe used … you need to know what you’re looking for
2. size of translocation/genetic change -small genetic variations are not detected by FISH

Question 6

what is Array-based comparative genomic hybridization?

(arrayCGH)

Answer 6

it is a moethod for the detection of aneuploidy and chormosomal copy number changes on a genome wide and high resolution scale

• it provides a rapid and cheap test to detect genetic changes that result in the gain or loss of DNA

Question 7

what is the benefit of using arrayCGH?

Answer 7

it can detect aneuploidy as well as submicroscoping rearrangements of DNA

capable of whole genome wide scan

less labour intesive (therefore cheaper)

Question 8

how do we perform an arrayCGH?

Answer 8

1. isolate genomic DNA from patient/test sample
2. DNA digestion
3. flourescently label patient/test and control/reference samples
4. hybridize to microarray
5. post hybridization waching
6. assay scanning and data analysis

Question 9

what are the limitations of assayCGH?

Answer 9

does not detect balanced translocations

distinguishing between normal variants and pathologically significant variants can sometimes be challenging