Chapter 7

Question 1

what is the definition of hybridization?

Answer 1

hybridization

> the process of forming an double stranded nucleic acid from joining two complementary (single) strands of DNA (or RNA)

Question 2

what is denaturing?

Answer 2

heating of DNA -> the two DNA strands separate

Question 3

what are the steps when forming a probe-target heteroduplex?

Answer 3

probe-target:

1. sampe: contains heterogenous nucleic acid fragments, including target
2. probe: known nucleic acid sequences

>>> denature both and mix

results:

1. re-annealed sample (homoduplex)
2. re-annealed probe (homoduplex)
3. probe-target heteroduplex

Question 4

what are two ways to label probes?

Answer 4

1. radioisotopes
2. fluorophobes

Question 5

what are 2 disadvantages of radioisotopes?

Answer 5

1. health hazard
2. radioactive decay

> necessary to synthesize fresh probes before each experiment

Question 6

how do fluorophobes work?

Answer 6

fluorophobes

> chemical group that can be detected because it absorbs energy of a specific wavelength and then re-emmits it at a specific but longer wavelength

Question 7

4 steps in souther blot hybridization

Answer 7

southern blot hybridization

1. digest DNA with restriction endonucleases and separate fragments by size on an agarose gel
2. denature DNA and transfer to membrane
3. hybridize labeled probe to DNA fragments bound to membrane
4. wash to remove unhybridized probes and apply X-ray film

Question 8

what is a northern blot?

Answer 8

a variation of a southern blot, but then with RNA samples

> can provide information on the range of cell types in which a certain gene is expressed

Question 9

what is chromosome in-situ hybridization?

Answer 9

e.g. whole-chromosome painting

> probe anneals to large parts of a specific chromosome

Question 10

DNA of human chromosome 2 was labeled and applied to orang-utan and human metaphse chr.

> conclusion?

Answer 10

orang-utans carry 2 sets of chromosomes with orthologous DNA sequences for human chr 2

> human chr 2 is a fusion of 2 chromosomes (happened after split of human/chimpanzee lineages

Question 11

how big is a genechip array?

Answer 11

1.28 x 1.28 cm

Question 12

how many locations on one genechip array?

how many DNA strands in one location?

Answer 12

500.000 locations on one chip

millions of DNA strands in one location

Question 13

studying transccriptome with miroarrays

> how?

Answer 13

1. microarray: many thousands unlabeled DNA or probe populations fixed to a surface at precisely defined coordinates
2. test sample (denatured and labelled) is hybridized to the probe molecules on the microaray
3. wash - dry > labels are detected with high resolution laser scanner and signal from each spot on the microarray is analyzed with digital imaging software

Question 14

what are paralogous genes?

Answer 14

paralogous genes

> two genes of clusters of genes at different chromosomal locations in the same organism that have structural similarities indicating that they derived from a common acestral gne and have singe diverged from the parent copy by mutation and selection of drift

Question 15

what is alternative splicing?

Answer 15

alternative splicing:

> results in different combinations of exons becoming linked together , resulting in different proteins being synthesized from the same pre-mRNA

Question 16

what is a problem when using microarrays in combination with mRNA samples?

Answer 16

microarray complication

> insufficient specifity to distinguish between every mRNA that is present

> due to paralogous genes/alternative splicing