chapter 5 Exploring genes and genome

Question 1

southern blotting used for

Answer 1

DNA

Question 2

restriction enzymes are

Answer 2

restriction endonucleases that cleave both strands of the recognised DNA

Question 3

characteristic of cleavage sites when talking about RE

Answer 3

twofold rotational symmetry

Question 4

when is polyacrylamide gel is used

what about more porous agarose gel

Answer 4

1000 bp

larger fragments as large as 20kbp

Question 5

how to mmake sure that the chain termination in sanger sequencing will only take place occasionaly

Answer 5

by making the concentration of the deoxy analog very low

Question 6

number of bp that can be sequenced using sanger sequencing

Answer 6

1000 bp

Question 7

what is stringency in PCR how can it be controlled?

Answer 7

the required closeness of the match between the primer and target . by adjusting temperature and salt

Question 8

when is the DNA only between primers amplified

Answer 8

at high temperatures

Question 9

RFLPs

Answer 9

(restriction fragment length polymorphisms) polymorphisms within restriction sites that change the size of DNA produced using apropriate enzymes

Question 10

what sources of energy does a ligase in RE use?

Answer 10

ATP or NAD+

Question 11

how is the problem with not having restriction sites in DNA solved?

Answer 11

Solved by adding chemically synthesized DNA-linkers

Question 12

what do plasmids carry?

Answer 12

antibiotics inactivating genes
toxin production
natural products degrading genes

Question 13

polylinker region?

Answer 13

has many unique restriction sites within its sequence

Question 14

Reporter genes

Answer 14

plasmids, easily detectable markers such as antibiotic resisting enzymes

Question 15

expression vectors

Answer 15

plasmids for the production of large amounts of protein

Question 16

Advantages of using modified vira

Answer 16

they enter bacteria much more easily than do plasmids

Question 17

cosmids

Answer 17

hybrids of lambda phage and a plasmid used for the insertion of large DNA-inserts

Question 18

what agent is used to denature DNA for DNA hybridisation

Answer 18

sodium hydroxide

Question 19

which one is resistant to alkaline hydrolysis DNA or RNA

Answer 19

DNA

Question 20

what enzyme is used for synthesizing the second strand when RNA-strand is hydrolysed in cDNA synthesis and what does it do

Answer 20

terminal transferase
adds G bases to the same strand
and then a primer of C-nucleotides is added to the newly synthesized complementary strand

Question 21

The principle behind site-directed mutagenesis

Answer 21

using primers with one mismatch

Question 22

inverse PCR why used and mechanism

Answer 22

used to for example introduce deletions, by using primers that orient in different directions to the ones used in normal
PCR

Question 23

mobile genetic elements

Answer 23

non-coding DNA in the human genome, from retrovira inserted themselves over time.

Question 24

example of mobile genetic elements in the non-coding DNA in the human genome

Answer 24

SINES (short interspersed elements)

LINES (long interspersed elements)

Question 25

In pyrosequencing light is produced when

Answer 25

the correct nucleotide is incorporated

Question 26

the reactions in pyrosequencing

Answer 26

1. ppi+ adenylylsulfate = ATP + sullfate

2- ATP + luciferin –> oxyluciferin + light

Question 27

enzymes used in pyrosequencing

Answer 27

sulfurylase
luciferase
apyrase

Question 28

how can the quantity of individual mRNA be determined

Answer 28

by using qPCR (quantitative PCR) or real PCR

Question 29

CT (the cycle number in which the fluorescence is detectable)

Answer 29

inversely proportional to the number of copies

Question 30

The relationship between CT and starting quantity

Answer 30

linear

Question 31

how is DNA introduced to animal cells

Answer 31

1. precipitation using calcium phosphate incorporation not efficient
2. Retroviruses
3. glass micropipetttes

Question 32

homologous recombination

Answer 32

Two DNA segments have very strong similarity exchange certain segments

Question 33

Repairing double strand breaks in cells

Answer 33

No template => non-homologous end joining

Question 34

Homology directed repair HDR

Answer 34

repair using a template

Question 35

Cas 9

Answer 35

2 lobe
REC for the recognition of DNA+sgRNA

NUC for the cleavage of both strands

Question 36

PAM

Answer 36

Protospacer adjacent motif

Question 37

gene knocknout

Answer 37

the expression of a gene is completely eliminated

Question 38

gene knockdown

Answer 38

the expression of a gene is reduced but not eliminated

Question 39

RISC

Answer 39

RNA induced silencing complex cleaves the passenger strand. siRNA