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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
What does Palindromic mean?
Its when the sequences is the same as complimentary sequences in opposite direction
26
What do REs bind as?
Homodimers
27
A few REs binds as heterodimers and are recognized as?
asymmetric sequences
28
What cuts specific cut to RE make?
cuts phosphodiester bonds
29
What are the ends called when the fragment results in a CLEAN CUT?
Blunt ends
30
Which direciton do blunt cuts cut in?
clean cutt up and down
31
What are the ends called when the fragment results in a staggered cut?
Sticky ends
32
Can sticky ends by ligated (spliced together)
yes if complimentary
33
How many modification systems do REs have?
1-2, typically DNA methyltransferases
34
What can be added within the recognition sequence?
Methyl groups (CH3) which is added to a C or A
35
Methylated DNA is?
Protected
36
What does the methyl group extend to?
extends into the major groove of DNA sterically
37
What dos methylated DNA do?
blocks the enzymes from cutting DNA
38
Type 1 , 3, 4 are___?
restriction and modifiation enzymes
39
Type 2 most have__?
sepaerate restriction and modification enymes
40
Some REs have?
Star activity
41
What do star activity enzymes do?
Cleave sequences that are similar but not identical to their recognition sequence under non-standard reaction conditions
42
What does the star enzyme activity depend on?
depends on specific "new" conditions
43
What can the varied star activity lead to in a lab setting?
too much glycerol, ions other than Mg2+ in buffer, the wrong buffer, prolonged reaction time, too much enzymes
44
Why make rDNA?
So scientist can test their "gene of interest" in an expression system and to produce a protein (ex. insulin)
45
In order to put your ___ into a model system, it must be put into a ___
Gel, vector
46
What is a vector?
A mode of delivering into an expression system
47
What is another source for a model system?
DNA and often plasmid but can it be viral or other resources as well?
48
Steps for making rDNA?
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
What does DNA ligase do?
Joins DNA fragments which allows creation of rDNA
50
Do sticky ends, or blunt ends ligate more efficiently?
Sticky ends
51
Not all ligase can join__
blunt ends
52
____ is the preferred ligase use in laboratories
T4 DNA ligase
53
rDNA can cut with __
1 enzyme; problem can go either way
54
Why is it easy for plasmi to lcose back on itself?
Because it closed back without accepting t insert
55
If rDNA cuts with 2 enzymes, what ligation can occur?
directional, which only way for the insert to fit
56
How do you get only the bands you want to build rDNA?
Gel purification, cut out bans you want, then purify the DNA from excised gel
57
What is gel purification?
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
Why would scientists want only to get the bands to build rDNA?
1. diagnosis of an inherited disorder 2. paternity 3. crime scene investigation 4. helpful tool to research rDNA
59
How to set up restriction Digestion
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
What is the reaction from DNA?
clean amounts depends on purpose
61
what is the reaction from RE?
an enzyme to cut your DNA and correct volume
62
What is the reaction from buffer?
need the correct environment (pH, salt concentration, cofactors) specified for enzymes
63
What is the reaction from dH2O
to bring reaction to correct volume (to give correct concentrations)
64
What is a control reaction?
uncut DNA

Intro to Restriction Enzymes (1 decks)

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