DNA-based Methods of Deletion and Duplication Detection

Question 1

Small length mutations may be identified by ___ followed by ____.

Answer 1

PCR; DNA sequencing

Question 2

Larger changes in length can be detected by ____ or by one of a range of more modern techniques.

Answer 2

Southern blot analysis

Question 3

Large expansions of trinucleotide repeats, particularly in ____ and ____, can be detected by an alternative technique named ____.

Answer 3

Myotonic dystrophy; Friedreich’s ataxa; triplet repeat-primed PCR

Question 4

What is the trinucleotide repeat associated with myotonic dystrophy?

Answer 4

(CTG)n

Question 5

What is the trinucleotide repeat associated with Friedrieich’s ataxa?

Answer 5

(GAA)n

Question 6

What is the trinucleotide repeat associated with fragile X syndrome?

Answer 6

(CGG)n

Question 7

The large (CGG) repeat in fragile X syndrome is more difficult to analyse by using triplet repeat-primed PCR due to the resulting ___ and consequent abnormally high levels of ____ in this expansion, located in the ____ of the associated ____ gene

Answer 7

very high CG content; hydrogen bonding; promoter region of FMR1 gene.

Question 8

What method is used usually to detect Fragile X syndrome?

Answer 8

Southern blotting

Question 9

An increasingly used alternative method for the detection of large deletions and amplifications affecting specific genes is the technique known as ____ in which PCR is used to simultaneously analyse the copy number at multiple points along a particular DNA sequence.

Answer 9

multiplex ligation - dependent probe amplification (MLPA)

Question 10

A range of commercially developed MLPA kits is now in routine use in diagnostic laboratories for the rapid detection of deletions affecting the genes responsible for various conditions including?

Answer 10

hereditary breast and colorectal cancer
neurofibromatosis type 1
Williams syndrome
velocardiofacial syndrome
Prader – Willi syndrome
Angelman syndrome
Smith – Magenis syndrome

Question 11

Deletions and duplications associated with these diseases involve several exons.

Answer 11

Duchenne muscular dystrophy; some forms of cancer such as breast cancer, i.e., involves BRCA1 and BRCA 2

Question 12

In general, deletions and duplications involving several exons are difficult to detect using ____ and are likely to be too small to detect by ____ or even ____.

Answer 12

DNA sequencing; karyotyping; in-situ hybridization

Question 13

This method involves amplifying several regions of a gene simultaneously, allowing a subsequent comparison to be made of the abundance of each of the resulting products, thus revealing regions of altered copy number (duplications).

Answer 13

multiplex PCR

Question 14

This technique is particularly useful when the location of submicroscopic genomic deletions or duplications is not already suspected and when the phenotype suggests that such a deletion may be present even though standard karyotyping has been apparently normal.

Answer 14

array comparative genomic hybridization (aCGH)

Question 15

This technique of detecting submicroscopic genomic deletions or duplications involves comparing, at multiple defined positions across the genome, the abundance of an individual ’ s test DNA relative to the abundance of a reference DNA.

Answer 15

array comparative genomic hybridization (aCGH)

Question 16

Array comparative genomic hybridization is carried out by adding the fluorescently labelled subject ’ s DNA and the reference DNA to ____ that contain thousands of specific DNA sequences, then washing off the unbound DNA and laser scanning the ____.

Answer 16

microarrays

Question 17

T/F: DNA hybridization to microarrays can also be used to screen directly for the presence of point mutations in several genes simultaneously.

Answer 17

True (microarrays contain thousand of specific DNA sequences to which test DNA can hybridize)

Question 18

Microarrays can be used to check for a large number of possible mutations in the many genes that can cause _____ and _____.

Answer 18

Childhood deafness; cardiomyopathy

Question 19

T/F: The arrays generally have to be custom - designed and they are not used widely in diagnostic laboratories in the UK at present for this purpose.

Answer 19

True

Question 20

Specific regions of genomic DNA can be captured by hybridization to custom – designed oligonucleotide arrays in order that these selected DNA regions can subsequently undergo ____.

Answer 20

NGS

Question 21

_____ method of DNA enrichment such as array comparative hybridization can even be used to select for all protein - encoding exons prior to massively parallel sequencing.

Answer 21

Hybridization-mediated

Question 22

This method can be used to detect large changes in the length of a region of DNA, for instance those resulting from a large deletion or duplication. Now used much less.

Answer 22

Southern blot

Question 23

Deletions may be evident as ____ or bands of ____ in agarose gel.

Answer 23

absent bands; bands of reduced molecular weight

Question 24

In Southern blot, DNA is cleaved into fragments using a specific ____ and the fragments are then separated according to size by ____. These fragments are then transferred to a ____ and the fragments of interest are identified using a specific ____.

Answer 24

restriction enzyme; gel electrophoresis; DNA-binding filter; DNA probe

Question 25

In Southern blotting, ____ are sections of DNA, ranging from tens of base pairs to several kilobases (kb) in size, w/c are used to identify complementary base sequences.

Answer 25

DNA probes

Question 26

In Southern blot, probe labelling is achieved by the incorporation of modified nucleotides that are either ____ or, alternatively, have an attached molecule that can subsequently be used to trigger____ that emits light

Answer 26

radioactive; chemiluminescent enzymatic reaction

Question 27

Give one example of an enzyme used in DNA probes for Southern blot.

Answer 27

Digoxigenin

Question 28

The probe and its (filter - bound) target sequences are firstly ____ to render them single - stranded and then incubated together.

Answer 28

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