Lecture 5 Restriction Enzymes and Bioinformatics

Question 1

Restriction Endonucleases

Answer 1

Restriction enzymes permit hydrolysis of phosphodiester backbone of DS-DNA (only cut double stranded DNA, not RNA or protein)

Type II RE most often used -> endonucleases that cleave at specific recognition site (somewhere in the middle)

Type I and III are not used since it creates problems when cut far away from recognition sites:
- Might include unwanted or unnecessary DNA
- If you cut randomly, you can’t predict what you might see

Question 2

What is difference between blunt ends and sticky ends?

Answer 2

Twofold symmetry: point of cleavage is within axis of symmetry

Blunt ends: cleave both strands exactly at the same axis

Sticky ends: cleave each strand at similar locations on opposite sides of the axis of symmetry

Question 3

Types of restriction digests

Answer 3

Sticky Ends:
5’ protruding ends (staggered with 5’ ends overhang)
3’ protruding ends (3’ sticky ends)

Blunt Ends:
No overhang

Question 4

Which has a higher efficiency when carrying out cloning?

Answer 4

Sticky ends since they can form complementary base pairs and join together
Blunt ends can’t form cbp and it needs to join in between which is less efficient

Question 5

What are the types of dye that can be used to visualize fragment?

Answer 5

DNA fragments can be visualized using gel electrophoresis by staining the DNA with:
1. Ethidium bromide or DNA ‘Safe’ Stain (SYBR-Safe) which goes in between and binds to the DNA strands
2. Fragments of nucleic acid are visible with UV light
3. Methylene blue is visible under white light and not a carcinogen (not as sensitive)

Question 6

Reaction buffers needed for enzyme activity

Answer 6

• Source of divalent cations (MgCl2) since Mg2+ is required as a cofactor for most enzymes
• NaCl or KCl (optimal ionic strength)
• Tris/HCl Buffer to maintain pH (ideal 7.2-7.6)
• Universal buffer works for all buffers

Question 7

What are ideal conditions to digest?

Answer 7

Temperature - most often 37C
Incubation time - time to digest the DNA ~ 1-2 hrs

Question 8

Why are enzymes stored in 50% glycerol?

Answer 8

They are stored in 50% glycerol at -20C to prevent freezing of the enzyme

The final concentration of glycerol must be <5% to function in an enzyme digest

Glycerol keeps the enzyme stable for long periods of time

Question 9

What a restriction enzyme unit?

Answer 9

One unit of restriction endonuclease is defined as the amt of enzyme required to digest 1 microgram of substrate DNA in 60 mins

Stock = 50 U/uL

Question 10

How to stop restriction enzyme reactions?

Answer 10

RE reactions can be stopped by adding a viscous solution (glycerol, bromophenol blue, EDTA chelates cations and stops enzyme action)

Under buffer conditions (pH 8.3) used DNA carries a slight negative charge and runs from negative (cathode) to positive pole (anode)

Question 11

What is starring in restriction digest?

Answer 11

Starring is digestion at sites other than its recognition sequence

If incorrect buffer is used or have high glycerol conc. and random cutting on DNA, it can lead to starring

Question 12

What direction does DNA migrate on gel?

Answer 12

Since DNA is negatively charged, it will migrate from negative (cathode) to positive pole (anode)

Question 13

What are forms of plasmid on a gel?

Answer 13

1. Natural form is supercoiled (want large amount in plasmid prep) - high mobility, travel furthest
2. Linear if nicked in two places (larger amounts in prep indicates poor technique) - moderate mobility
3. Open circle if nicked on one strand (common in plasmid prep) - low mobility, slowest

Need a break in both strands for DNA to become linear
More surface area, slow it travels

Question 14

What is restriction mapping?

Answer 14

A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes.

Method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints.

Question 15

What is bioinforgraphics and why is it important as a tool?

Answer 15

Combination of biological data and computational analysis tools -> a number of diff applications

• Gene/protein function and sequence analysis
• Evolutionary relationships
• Drug design

Bioinformatics is a tool that can process tremendous amount of data that is generated and useful for:
- Genomes
- Transcriptome (collection of all transcripts in the cell)
- Metabolome