2.2. NUCLEIC ACID HYBRIDISATION-BASED TECHNIQUES

Question 1

Hybridization

Answer 1

Establishment of a sequence- specific interaction between two complementary nucleic acid strands

Question 2

Strand dissociation

Answer 2

Denaturation, melting

Question 3

Strand association

Answer 3

Renaturation, annealing

Question 4

FISH

Answer 4

1. Fluorescence in situation hybridization
2. Molecular cytogenic (chromosome level) technique that uses fluorescent signal & nucleic acid hybridization to detect a sequence of interest

Question 5

Hapten

Answer 5

Small molecules against which an antibody can be raised

Question 6

Probes

Answer 6

1. Complementary sequences of nucleic acids
2. Designed to hybridize to the sequence of interest
3. Tagged with fluorescent dyes

Question 7

Locus specific probes

Answer 7

1. Bind to a particular chromosomal region

2. Determine on which chromosome a particular gene is located

Question 8

PCR

Answer 8

1. Polymerase chain reaction
2. Used to amplify a short, well-defined part of a DNA strand
   - > single gene or part of a gene

Question 9

Denaturation

Answer 9

(95oC)
1. Double-stranded DNA ‘melts’ open by disrupting the hydrogen bonds between complementary bases to form single-stranded DNA

Question 10

Annealing/ hybridization

Answer 10

(~55oC)

Primer anneals to the single stranded DNA

Question 11

Extension/ elongation

Answer 11

(72oC)

1. Polymerase adds deoxynucleoside triphosphate in a template-dependent fashion

Question 12

dNTPs

Answer 12

Deoxynucleoside triphosphates

Question 13

Taq. Polymerase

Answer 13

1. Thermus aquaticus

2. Thermostable DNA polymerase

Question 14

Sequence specific

Answer 14

Primer length determines specificity

Question 15

Electrophoresis

Answer 15

1. Gel electrophoresis
2. The motion of charged particles in a fluid under the influence of an electric field
3. Migration through gel separates molecules by size

Question 16

Amplicons

Answer 16

PCR products

Question 17

RT-PCR

Answer 17

1. Reverse transcriptase PCR
2. Uses RNA as the template
   - with RNA-directed DNA polymerase (rTh)
3. Product is double-stranded cDNA

Question 18

Restriction endonucleases

Answer 18

1. Restriction enzyme
2. Molecular scissors
   - > cut double-stranded DNA at specific sequences

Question 19

Cytogenetic

Answer 19

Chromosome level