lecture 1. 7/18

Question 1

what dna technology rely on?

Answer 1

hybridization

Question 2

hybidization assays main principle?

Answer 2

to dectect a dna molecule (target) within a complex mixture.

Question 3

what is a probe?

Answer 3

dna sequence of knwon sequence. could be a dna sequence of a mutated gene.

Question 4

why are probes labeled?

Answer 4

to be able to see. usually radioactive

Question 5

main thing a probe must have?

Answer 5

complementary to the target sequence

Question 6

what are the 3 possible outcomes of probing with target dna?

Answer 6

1. target dna ca recombine
2. probe can rennealed
3. probe-target heteroduplexes. (best scenario)

Question 7

stringency?

Answer 7

the degree to which non-complementary sequences are tolerated during hybridization.

Question 8

the higher the stringency? what happens?

Answer 8

the fewer mismatches will be tolerated

Question 9

what are the hybridization of the target gene and probe an example of?

Answer 9

orthologs or paralogs.

Question 10

factors that correlates with stringency or decrease it?

Answer 10

salt concentration,
temperature
denature agents

Question 11

different ways to make hybridization?

Answer 11

dna probe with dna target
dna probe with rna targe
rna probe with rna targets

Question 12

Northern blotting mainly used for what?

Answer 12

to measure gene expression. look at rna sequences

Question 13

western blotting used for what

Answer 13

to id proteins with antibodies after electrophoresis and electro-transfer. not used for diagnosis.

Question 14

why to diagnosis is preferable to look into dna and not proteins?

Answer 14

hard to analyse proteins than it is to analyse dna. proteins can be sequenced but it is much harder. dna is much easier to do.
practical reason: much easier to get a dna sample because it can be collected anywhere in the body. to get a protein sample need to look at specific tissue that expresses it like the brain for huntingtons disease.

Question 15

how southern blotting works?

Answer 15

target will be genomic dna (chormosomes)
not the whole chromosome, digest it with restrictive enzymes.
separate thorugh gel electrophoresis.
take a section of a similar size section from the gel and then do the hybridization with the probes.
Main feature of Southern: transfer dna to membrane.
put membrane in a bag, add buffer and probe, set temp. and allow hybridization.
wash membrane, take memebrane to x-ray and expose to film and develop it.

Question 16

what the bands on the southern membrane represent?

Answer 16

correspond to size of segments.

Question 17

would you expect to see differences between tissues in southern blots from a single patients?

Answer 17

no, bc it is genomic dnd, wont change from tissue to tissue

*in northern blotting (protein analysis) difference would be seen from tissues to tissues

Question 18

polymorphism?

Answer 18

difference in sequences of alleles common in the population

Question 19

what is restriction fragment length polymorphism? RFLP

Answer 19

a polymorphosm that results in change in the size of a restriction fragment

Question 20

what are RFLP mainly used for?

Answer 20

look at different alleles

Question 21

what type of sequence is used to id different alleles?

Answer 21

restriction site where restriction sites can cut

Question 22

what is the meaning of the place where the probe binds?

Answer 22

where you can see in the southern blot

Question 23

what is the banding pattern indicate in the southern blot?

Answer 23

different alleles both bind to the different blotting

Question 24

what is the marker for the mutation on the allele?

Answer 24

the ??????

Question 25

why are some fragments not shown in southern blotting?

Answer 25

bc only fragments that bound to the probe will show on membrane

Question 26

what is the disadvantages of using rflp?

Answer 26

the fact of having only 2 alleles, the restriction sites are either present of not.

Question 27

how can restriction fragments also change?

Answer 27

chromosomeal rearrangememts

deletion or insertion bwt 2 restriction sites

Question 28

what is the main cause of hemophilia?

Answer 28

defects in the genes that is involved in the blood clotting cascade.

Question 29

hemophilia A?

Answer 29

affects 1 -4000 males at birth. coagulation disorder that cause prolonged bleeding times, easy bleeding and hemorrhage into joints and muscles

Question 30

how presentation of disease correlate with mutation?

Answer 30

different protein function activity

Question 31

problem with repetitive sequnce?

Answer 31

they undergo recombination duting miosis, and they can pair up and recombinate and crossover. can lead to inversion , deletion or insertions.