Nucleic Acid Hybridization

Question 1

Hybridization

Answer 1

Depends on complementary base pairing when the denatured ss probe is added to denatured nucleic acid sequences

Question 2

Affecting factors

Answer 2

Probe length, % of gc bps, conc of salt and chemical denaturants

Question 3

Hybridization steps

Answer 3

Incubate denatured probe with target, wash at high stringency, then the results are processed on a membrane

Question 4

Stringency

Answer 4

The level of base complementarity required to form stable hybrids

Question 5

How to increase stringency?

Answer 5

By increasing temp and decreasing salt conc

Question 6

Visualizing probes

Answer 6

Adding a chemically labelled or fluorescently labeled base, or swapping the phosphate with a radioactive one

Question 7

Colony hybridization

Answer 7

Screening of library to find the colony that has the gene of interest

Question 8

Genomic library/cDNA library

Answer 8

Collection of living bacterial colonies that have been transformed with different pieces of DNA from the organism of interest

Question 9

cDNA

Answer 9

Synthetic copy of all RNA in cell, it represents the transcriptome

Question 10

gDNA

Answer 10

All of chromosomal DNA in genome, it represents the genome

Question 11

Screening of genomic clones

Answer 11

Not expressed by bacteria so nucleic acid hybridization is used

Question 12

Screening of cDNA clones

Answer 12

Expressed by bacteria when put into an expression vector which is detected by an antibody

Question 13

Gel used for electrophoresis

Answer 13

Agarose gel immersed in a buffer solution

Question 14

Types of buffer solutions

Answer 14

Tris acetate EDTA, tris borate EDTA, 10mM sodium borate, 5mM lithium acetate

Question 15

Difference between southern and northern blotting

Answer 15

DNA is the target in southern while RNA is the target in northern

Question 16

Process of southern and northern blotting

Answer 16

Electrophoresis to separate DNA fragments, transfer them to a nitro cellulose membrane using capillary flow, hybridization with labelled probe

Question 17

Applications of southern blotting

Answer 17

Detecting polymorphism in DNA and detecting presence of DNA sequence

Question 18

Applications of northern blotting

Answer 18

Determine if gene is being expressed or not, reveal sizes of RNA

Question 19

Chromosome in situ hybridization

Answer 19

Metaphase chromosomes probed with DNA that are labelled with a fluorochome

Question 20

Tissue in situ hybridization

Answer 20

Sliced or whole mounted preparations probed with RNA to detect mRNA expression

Question 21

Western blotting

Answer 21

Used to detect specific protein molecules from a mixture of proteins