Techniques for analysing nucleic acids

Question 1

Techniques for analysing nucleic acids

Answer 1

Restriction enzyme (RE) mapping
Gel electrophoresis
Nucleic acid blotting
In situ hybridisation

Question 2

Restriction enzyme mapping

Answer 2

• relies on using palindromic recognition sites to positionally locate RE sites on a piece of DA
• Combination of the number, order and distance between RE sites in a DNA fragment
• Fragments generated with RE digestion

Question 3

Restriction enzyme Mapping units

Answer 3

– bp or kb

Question 4

RE Mapping | can provide information about

Answer 4

1. length of a cloned fragment
2. Position of RE sites for subcloning
   3.Comparison of internal organization

Question 5

Slide 9. Chapt.20 - Part 2

Question 6

Size determination by

Answer 6

gel electrophoresis

Question 7

Bioinformatics

Answer 7

– now restriction maps are generated using software that identify RE sites in a DNA fragment, based on the nucleotide sequence

Question 8

RE mapping for:

Answer 8

Limited characterisation of DNA fragments about which little is known beforehand
Can serve as starting point for isolation of a gene
Refine genetic maps (RE sites serve as markers)
Diagnostic if a restriction site lies close to a mutant allele (RFLP)

Question 9

Gel electrophoresis

Answer 9

Molecular sieve
- Agarose (DNA/RNA)
- Polyacrylamide (Proteins)
Electric charge (DNA is –ve charged)
Separation by fragment size (markers)
Sizes judged by comparing with known size markers (DNA ladder)
Visualisation with EtBr or SYBR Green using UV light

Question 10

Question 11

Nucleic Acid Blotting

Answer 11

[relies on hybridization between complementary nucleic acid molecules (DNA or RNA).]
Immobilising DNA on solid supports (nitrocellulose or nylon membrane) prior to hybridisation with labelled probe
Thus: relies on hybridisation of two complementary ss-DNA fragments
Southern blot (Edwin Southern – DNA – 1975)
Northern (RNA) [& Western (protein)]
Probes
> DNA/RNA – DNA/RNA
> [Proteins – antibodies]

Question 12

The Southern blot

Answer 12

DNA cut with RE
Agarose gel electrophoresis
Denature DNA in gel (ss)
Transfer to membrane (capillary action)
Hybridise with probe (DNA/RNA)
Hybrid forms between denatured probe and homologous nylon-bound ssDNA.
Unbound DNA washed off
Visualised by autoradiography (radioactivity or chemiluminescense)

Question 13

Southern blot

Question 14

Nucleic acid hybridization:

Answer 14

A technique in which single-stranded nucleic acids (DNA or RNA) are allowed to interact so that complexes called hybrids are formed by molecules with similar, complementary sequences.

Question 15

Nucleic Acid Blotting relies on

Answer 15

hybridization between complementary nucleic acid molecules (DNA or RNA).

Question 16

A probe

Answer 16

is a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome. … The probe is labeled with a radioactive or chemical tag that allows its binding to be visualized.

Question 17

Question 18

The southern blot deals exclusively with _____, nothern blotting deals exclusively with_____

Answer 18

DNA
RNA

Question 19

Prior to the development of RT-PCR and real-time PCR, ____________ was commonly used to study gene expression.

Answer 19

Northern blotting

Question 20

Northern blotting

Answer 20

mRNA extracted from tissue
Separated by gel electrophoresis
Transferred to membrane, hybridised with ss-DNA/RNA probe
Identify complementary mRNA species

Question 21

Slide 13. Chapt 20 - Part 2

Question 22

In a Northern blot, the membrane is exposed to a labeled single-stranded DNA or RNA probe derived from a cloned copy of the gene of interest. If mRNA complementary to the probe is present, the complementary sequences will hybridize and be detected.

Question 23

Flourescence in situ Hybridisation (FISH)

Answer 23

hybridizes a probe to DNA or RNA without blotting.

Question 24

Fluorescence in situ Hybridisation (FISH) use _________________, particularly when embryos are used for various studies in developmental genetics

Answer 24

tissue sections or entire organisms

Question 25

Consider an in situ hybridization as a spatio-temporal view of the abundance of nucleic acid in your chosen study model. Unlike a Northern blot, it gives you an idea of

Answer 25

where (tissue specificity) and when (time) a gene is expressed in your model system.

Question 26

In the example in situ hybridization of a zebrafish embryo 48 h after fertilization. 2. showing expression of mRNA (probe results in dark blue color) 3. The mRNA is restricted to muscle cells surrounding the developing spinal cord of the embryo.