L9 - molecular bio 3 - nucleic acid probes and hybridization

Question 1

what is nucleic acid hybridization

Answer 1

method to identify nucleic acid clones/fragments containing specific sequences

Question 2

what can separate a DNA strand

Answer 2

heat

alkali conditions

Question 3

define hybridize

Answer 3

complimentary strands will join up (hybridize)

Question 4

can DNA and RNA hybridize to ssDNA

Answer 4

yes

Question 5

why do we carry out nuclear acid hybridization technique?

Answer 5

may have a sequence of DNA and we want to see what plasmids in the cDNA have this sequence

Question 6

how is the DNA probe made

for hybridization

Answer 6

1. denature template and add primer
2. add DNA pol and radiolabelled ddNTPs for it to incorporate along from the primer
3. denature again to free the labelled primer

Question 7

what is the name of the modified DNA pol used in process of making hybridization probe

Answer 7

klenow fragment

Question 8

describe process of colony blot hybridization

Answer 8

1. bacterial colonies (containing cDNA plasmids) grown then transferred onto DNA binding material
2. bacteria lysed and denatured using alkali
3. place in solution with labelled probe
4. complimentary sequences will hybridize
5. rinse and expose to x-ray film to identify radiolabels
6. separate hybridised colonies and grow

Question 9

example of DNA binding material

Answer 9

nylon

nitrocellulose

Question 10

when is colony blot hybridization used

Answer 10

to compare probe to cDNA library

Question 11

when is southern blot hybridization used

Answer 11

we have an isolated sequence of DNA

we want to see what other DNA contains this sequence

Question 12

describe process of southern blot

Answer 12

1. digest DNA with RE
2. separate fragments by Gel electrophoresis
3. denature fragments in alkali
4. transfer to nitrocellulose/nylon
5. add labelled primer in solution with the ssDNA fragments
6. complimentary regions will hybridize and can be detected

Question 13

how are the ssDNA transferred from the gel to the DNA binding membrane?

Answer 13

this part is the actual southern blot method

membrane placed on top of gel with paper towels on top of membrane

paper towels suck the alkali solution up onto the DNA binding membrane

Question 14

when is northern blot used

Answer 14

when we have an isolated sequence of DNA
we want to see what RNA contains the sequence

eg to see if a gene (in DNA) is being expressed (in RNA)

Question 15

describe process of northern blot

Answer 15

1. mRNA extracted from sample and separated on agarose gel
2. transferred straight to membrane (no need to denature as ss)
3. labelled DNA probe added, will hybridize at complimentary sequences

Question 16

(in a northern blot) if our DNA probe doesn’t hybridize to any of the RNA what does this mean?

Answer 16

the gene from DNA isnt being expressed

Question 17

uses of northern blot

Answer 17

1. can compare expression (and extent of expression) of gene between tissues
2. can see how a disease affects gene expression
3. can see how gene expression changes over time (development)

Question 18

define in situ hybridization

Answer 18

hybridizing a probe directly to RNA without blotting onto membrane

can be carried out on tissue sections or entire organism

Question 19

advantages of in situ hybridisation

Answer 19

can use labelled probe to identify cellular and tissue distribution of mRNA (of specific genes)

Question 20

what are DNA microarrays

Answer 20

modern devices that use nucleic acid hybridization to rapidly measure which genes are present in a tissue sample (simultaneously)

study expression of a gene across entire genome

Question 21

how do DNA microarrays work

Answer 21

uses reverse approach

1. all mRNA converted to cDNA, fluorescently labelled and used as probe
2. probe applied to array which contains spots where ssDNA from each gene in genome is laid out
3. labelled cDNA probe will bind complimentarily and their fluorescence will be detected by chip reader

Question 22

what does brightness of spots in a microarray indicate?

Answer 22

extent of expression of a gene

Question 23

define transcriptome

Answer 23

set of transcripts in a cell/tissue/sample

Question 24

define transcriptomics

Answer 24

the study of transcriptomes

Question 25

what is comparative DNA microarrays

Answer 25

having 2+ samples, labelling different colours and hybridizing them to the same chip

eg sample 1 red
sample 2 green

genes expressed in both - yellow
genes expressed in only one sample : green/red