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Intro to Restriction Enzymes > Restriction enzymes > Flashcards

Restriction enzymes Flashcards

1
Q

What is restriction enzymes known as

A

Endonucleases=within, nuclease= cleaves DNA

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2
Q

What are restriction enzymes

A

Proteins that cut double-stranded DNA

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3
Q

How many incisions can restrictions enzymes make?

A

2 incisions

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4
Q

Where is the incision made?

A

One through each sugar-phosphate backbone of the double helix without damaging the bases

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5
Q

What can the bonds be reformed with?

A

DNA Ligase

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6
Q

How is Recombinant DNA (rDNA) created?

A

When fragments of different organisms or genes can be spliced (join together) if they “fit”

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7
Q

What are Restriction enzymes made out of?

A

Made out of bacteria and archaea as a defense mechanism

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8
Q

What does the defense mechanism in Restriction enzymes do?

A

Damage foreign DNA from viruses

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9
Q

How many typs of restrictions eneyes are their?

A

4

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10
Q

What are the 4 types of restriction enzymes based on?

A

Based on the complexity of the enzyme, cutting pattern, sequence, specificity, etc.

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11
Q

What does type 1 RE do?

A

A complex cut of DNA at random far from the recognition site

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12
Q

What does type 2 RE do?

A

(Most common type) the only type used in labs, cleaves DNA close to or within their recognition site, creating discrete fragments and distinct banding patterns.

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13
Q

How many recognition sequences are in type 2 RE?

A

4-8 bases in length

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14
Q

Do sub groups of type 2 exist?

A

yes

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15
Q

What does type 3 of RE do?

A

Cut outside of recognition site, rarely produce complete digest, similar to type 1 but less complex.

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16
Q

How many recognition sequences does type 3 require, and in which direction do those sequences face?

A

2 recognition sequences, and in opposite directions

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17
Q

What does type 4 of RE do?

A

Can recognize modified DNA, such as methylate DNA

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18
Q

What does type 4 of RE do?

A

Can recognize modified DNA, such as methylate DNA

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19
Q

What does the E stand for an RE?

A

Escherichia (genus)

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20
Q

What does the C stand for an RE?

A

coli (species)

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21
Q

What does the R stand for an RE?

A

RYI 3 (stain)

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22
Q

What does the I stand for an RE?

A

1st identified enzyme in that stain

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23
Q

What specific cut based on sequences of DNA is called?

A

Recognition sequences site

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24
Q

Most recognition sequences are?

A

Palindromic

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25
Q

What does Palindromic mean?

A

Its when the sequences is the same as complimentary sequences in opposite direction

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26
Q

What do REs bind as?

A

Homodimers

27
Q

A few REs binds as heterodimers and are recognized as?

A

asymmetric sequences

28
Q

What cuts specific cut to RE make?

A

cuts phosphodiester bonds

29
Q

What are the ends called when the fragment results in a CLEAN CUT?

A

Blunt ends

30
Q

Which direciton do blunt cuts cut in?

A

clean cutt up and down

31
Q

What are the ends called when the fragment results in a staggered cut?

A

Sticky ends

32
Q

Can sticky ends by ligated (spliced together)

A

yes if complimentary

33
Q

How many modification systems do REs have?

A

1-2, typically DNA methyltransferases

34
Q

What can be added within the recognition sequence?

A

Methyl groups (CH3) which is added to a C or A

35
Q

Methylated DNA is?

A

Protected

36
Q

What does the methyl group extend to?

A

extends into the major groove of DNA sterically

37
Q

What dos methylated DNA do?

A

blocks the enzymes from cutting DNA

38
Q

Type 1 , 3, 4 are___?

A

restriction and modifiation enzymes

39
Q

Type 2 most have__?

A

sepaerate restriction and modification enymes

40
Q

Some REs have?

A

Star activity

41
Q

What do star activity enzymes do?

A

Cleave sequences that are similar but not identical to their recognition sequence under non-standard reaction conditions

42
Q

What does the star enzyme activity depend on?

A

depends on specific “new” conditions

43
Q

What can the varied star activity lead to in a lab setting?

A

too much glycerol, ions other than Mg2+ in buffer, the wrong buffer, prolonged reaction time, too much enzymes

44
Q

Why make rDNA?

A

So scientist can test their “gene of interest” in an expression system and to produce a protein (ex. insulin)

45
Q

In order to put your ___ into a model system, it must be put into a ___

A

Gel, vector

46
Q

What is a vector?

A

A mode of delivering into an expression system

47
Q

What is another source for a model system?

A

DNA and often plasmid but can it be viral or other resources as well?

48
Q

Steps for making rDNA?

A

First, the vector must be ct o op plasmid (or another vector); done using restriction enzymes, you insert the gene of interest o cut the same restriction enzymes and then ligated to plasmid to creat rDNA

49
Q

What does DNA ligase do?

A

Joins DNA fragments which allows creation of rDNA

50
Q

Do sticky ends, or blunt ends ligate more efficiently?

A

Sticky ends

51
Q

Not all ligase can join__

A

blunt ends

52
Q

____ is the preferred ligase use in laboratories

A

T4 DNA ligase

53
Q

rDNA can cut with __

A

1 enzyme; problem can go either way

54
Q

Why is it easy for plasmi to lcose back on itself?

A

Because it closed back without accepting t insert

55
Q

If rDNA cuts with 2 enzymes, what ligation can occur?

A

directional, which only way for the insert to fit

56
Q

How do you get only the bands you want to build rDNA?

A

Gel purification, cut out bans you want, then purify the DNA from excised gel

57
Q

What is gel purification?

A

A technique that allows you to “run” the samples on an gel cut out bans you want, then purify the DNA from an excised gel.

58
Q

Why would scientists want only to get the bands to build rDNA?

A
  1. diagnosis of an inherited disorder
  2. paternity
  3. crime scene investigation
  4. helpful tool to research rDNA
59
Q

How to set up restriction Digestion

A

Keep enzymes on ice (in -20celcius freezer) because it becomes unstable at room temp; only want about 1ul of an enzyme, use 1 unit of enzyme will digest mg of DNA in a 50 ul reaction in 1 hr, an use up to 10x more enzyme for more efficient cleavage and short incubation time, mix tube, place in an incubator, or 20 min-1hr, control reaction

60
Q

What is the reaction from DNA?

A

clean amounts depends on purpose

61
Q

what is the reaction from RE?

A

an enzyme to cut your DNA and correct volume

62
Q

What is the reaction from buffer?

A

need the correct environment (pH, salt concentration, cofactors) specified for enzymes

63
Q

What is the reaction from dH2O

A

to bring reaction to correct volume (to give correct concentrations)

64
Q

What is a control reaction?

A

uncut DNA

Intro to Restriction Enzymes (1 decks)

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