Restriction enzymes and DNA cutting

Question 1

how is polyploidy genetic variation induced

Answer 1

need to add a chemical

Question 2

what is genetic engineering

Answer 2

manipulation of DNA of organisms for the purposes of, changing inheritance or the in vitro manufacture of gene products

Question 3

what is a mutation

Answer 3

any change in genetic information relative to reference ‘wild type’ genome

Question 4

what is a mutagen

Answer 4

any agent causing mutations

Question 5

how do mutations with mutagens occur

Answer 5

randomly

Question 6

what occurs in genetic engineering

Answer 6

enable us to take genes out
genetic material analysed
DNA can be altered
DNA can be re-inserted in organism

Question 7

how can DNA be cut

Answer 7

shaking DNA violently, but this will cause random breaking

DNA can be cut using enzymes in more specific and targeted way

Question 8

what are the two enzyme types

Answer 8

Endonucleases

Exonucleases

Question 9

what is an enzyme

Answer 9

a biological catalyst; all biochemical reactions are controlled by enzymes and in general, each enzyme controls each separate reaction

Question 10

what is a restriction enzyme

Answer 10

alternative name for endonuclease

Question 11

what is an exonuclease

Answer 11

cleaves nucleotides one at a time from the end (exo) of an polynucleotide chain

Question 12

what did Werner Arber discover

Answer 12

restriction enzymes

Question 13

what did Daniel Nathans discover

Answer 13

Pioneered the application of restriction for the construction of genetic maps

Question 14

what did Hamilton Smith discover

Answer 14

Showed that restriction enzyme cuts DNA in the middle of a specific sequence

Question 15

what happened in 1978

Answer 15

Arber, Nathans and Smith won a Nobel Prize

Question 16

how was HindlI discovered

Answer 16

is the first restriction enzyme discovered accidentally in 1970; while studying how the bacterium Haemophilus influenzae defends itself to the virus

Question 17

what can the bacteria Haemophilus influenzae do

Answer 17

break down the DNA from the phage and any other foreign DNA; yet its own DNA is not damaged

Question 18

what does base Y mean

Answer 18

C+T

Question 19

what does base R mean

Answer 19

G+A

Question 20

what can exonucleases be used for

Answer 20

• Removal of oligonucleotides before a PCR reaction
• Removal of chromsomal DNA in plasmid preparations
• Removal of DNA in RNA preparations
• Generation of single strand DNA (ssDNA) from linear double strand DNA (dsDNA)

Question 21

what is recombinant DNA technology

Answer 21

creation of a new combination of DNA segments that are not found together naturally

Question 22

what are the applications of recombinant DNA technology

Answer 22

Basic research on control of gene expression
Forensic medicine
Biotechnology - generate new products

Question 23

how do restriction endonucleases work

Answer 23

Cleave phosphodiester bonds of nucleic acids at an internal site
highly specific; can recognise 4 bases pair (HaeIII), 6bp(EcoRI), 8bp (NotI) or more
restriction sites that are recognised are usually palindromic

Question 24

what is the use of a restriction endonuclease

Answer 24

Cut DNA internally and in a more specific and targeted way; their role is vital for the use of Genetic Engineering

Question 25

what is a palindrome

Answer 25

a sequence of units that can be read the same way in either direction

Question 26

what is the frequency of enzyme recognition

Answer 26

4n | n = number of bases in recognition sequence

Question 27

if each position of the recognition site has 4 possible bases what is the probability of finding any one base

Answer 27

1/4

Question 28

where does Haelll recognise a segment

Answer 28

segment of 4 bases 5' GC/CC 3' 3' CC/GG 5'

Question 29

where does Smal recognise a segment

Answer 29

segment of 6 bases 5' CCC/GGG 3' 3' GGG/CCC 5'

Question 30

where does Notl recognise a segment

Answer 30

segment of 8 bases 5' GC/GGCCGC 3' 3' CGCCGG/CG 5'

Question 31

what are blunt ends

Answer 31

end of a DNA molecule where both stands ends in a base pair

Question 32

what is an overhang

Answer 32

stretch of unpaired nucleotides in the end of a DNA molecule, they can be in either strand

Question 33

what are sticky ends

Answer 33

overhangs of more than one nucleotide at the end of a DNA molecule; also called cohesive ends

Question 34

how are blunt ends generated

Answer 34

shearing DNA or by cutting with blunt end restriction endonucleases (eg. HindII or SmaI)

Question 35

what does deoxynucleotide terminal transferase do

Answer 35

add short poly-T sequences at the 3’-ends of an opened vector. Add short poly-A sequences to the 3’-ends of DNA to be cloned

Question 36

what is used to seal the blunt ends

Answer 36

ligase

Question 37

how is a sticky end formed

Answer 37

DNA strands not cut at the centre of the restriction site staggered cutting leaves overhanging segment of 4 base pairs 5' G| AA|TTC 3' 3' CTTAA| G 5'

Question 38

what do sticky ends form

Answer 38

transient double helical structures

Question 39

what is used to join sticky ends

Answer 39

ligase

Question 40

what happens if different DNA molecules are mixed

Answer 40

some ligation will generate new combinations

Question 41

which was the first enzyme discovered

Answer 41

Hindll

Question 42

which enzyme can create sticky or blunt ends

Answer 42

endonucleases

Question 43

what does a restriction map show

Answer 43

positions of restriction sites in sequence of DNA