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Enzymes with recognition sequences from 4 to 8 nucleotides in length each have uses in genetic engineering. 6-cutters (i.e. enzymes that have ...

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genetic engineering
Genetic Engineering
  • technology involved in removing, modifying, or adding genes to a DNA molecule
  • Aka recombinant DNA technology
slide2
Restriction endonucleases
  • Gel electrophoresis
  • Cloning vectors
  • Simple cloning exercise
nucleases
Nucleases
  • exonucleases
    • remove single nucleotides from 3'- or 5'-end depending on specificity
    • most exhibit specificity for either RNA, ssDNA or dsDNA
    • good for removing undesired nucleic acid or removing single stranded overhangs from dsDNA
  • endonucleases
    • cleaves phoshodiester bonds within fragments
  • lack of site specificity limits uses and reproducibility
slide5
Restriction enzymes are classified as endonucleases. Their biochemical activity is the hydrolysis ("digestion") of the phosphodiester backbone at specific sites in a DNA sequence. By "specific" we mean that an enzyme will only digest a DNA molecule after locating a particular sequence.
slide6
All restriction enzymes cut DNA between the 3’ carbon and the phosphate moiety of the phosphodiester bond.
origin and function
Origin and function
  • Bacterial origin = enzymes that cleave foreign DNA
  • Protect bacteria from bacteriophage infection
    • Restricts viral replication
  • Bacterium protects it’s own DNA by methylating those specific sequence motifs
slide8
Named after the organism from which they were derived
    • EcoRI from Escherichia coli
    • BamHI from Bacillus amyloliquefaciens
availability
Availability
  • Over 200 enzymes identified, many available commercially from biotechnology companies
slide10

Restriction Enzymes

  • site-specific endonucleases of prokaryotes
  • function to protect bacteria from phage (virus) infection
  • corresponding site-specific modifying enzyme (eg., methylase)
  • type II enzymes are powerful tools in molecular biology
restriction modification systems
Restriction/modification systems -

EcoRI restriction enzyme

EcoRI methylase

EcoRI

Meth.

EcoRI

classes
Classes
  • Type I
    • Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme
    • Therefore random/imprecise cuts
    • Not very useful for rDNA applications
restriction modification systems type iii
Restriction/modification systems – Type III
  • R-M systems type III (few examples)- Similar to type I- - Recognition sequence: 5-7 bp
  • - Cleavage site: 25-27 bp downstream of recognition site (enzyme moves DNA, helicase activity)
slide14
Type II
    • Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme
    • Used widely for molecular biology procedures
    • DNA sequence = symmetrical
      • Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence
restriction enzyme recognition sequences
Restriction Enzyme Recognition Sequences
  • The substrates for restriction enzymes are more-or-less specific sequences of double-stranded DNA called recognition sequences.
  • The length of restriction recognition sites varies
  • Length of the recognition sequence dictates how frequently the enzyme will cut in a random sequence of DNA.
a calculation to ponder
A calculation to ponder:
  • The enzyme Sau 3A1 cuts on the GATC sequence.
  • GATC is something that occurs by chance pretty frequently.
  • If a DNA sequence is evenly made up of G, A, T, and C nucleotides (i.e. 25% of each), we would expect to find the sequence “GATC" by chance about every 256 nucleotides on the average. Why is that? Because if we point to a nucleotide in a sequence at random, the chances would be one in four that it would be “G" (the first nucleotide in the recognition sequence). The chance that the next nucleotide is "A" is also 1 in 4; the chance that the nucleotide after that is "T" is 1 in 4; and the chance that the next one is “C" is also 1 in 4. Therefore, the chance that we have randomly pointed to a sequence that reads “GATC' is:
  • (1/4) x (1/4) x (1/4) x (1/4) = 1/256
slide17
Any recognition sequence that was four nucleotides in length could be found every 256 nucleotides (on the average) in this simple scenario. In actuality, sequences are usually not evenly made up of G, A, T, and C nucleotides, which skews the statistics a bit. In addition, certain short sequences may be more or less common in the DNA, which will also affect the frequency with which a recognition sequence is found. The dinucleotide CG is very uncommon in mammalian DNA, which makes it less likely that you will find a recognition sequence for the enzyme Hpa II (C^CGG).
  • Longer recognition sequences lead to lower probability of having a site at any point in a DNA strand.
slide18
Enzymes with recognition sequences from 4 to 8 nucleotides in length each have uses in genetic engineering. 6-cutters (i.e. enzymes that have recognition sequences specified by six nucleotides) are good for day-to-day cloning work: An example of a 6-cutter is HindIII (A^AGCTT) which cuts the genome of bacteriophage lambda (48 kbp) at 7 sites.
slide19
8-cutters are good for carving up chromosomes into specific pieces that are still quite large. An example of an 8-cutter is NotI (GC^GGCCGC) - the NotI recognition sequence is not present in the genome of bacteriophage lambda.4-cutters are good for experiments where you want the possibility of cleavage at many potential sites. There are 116 Sau3AI sites in the genome of bacteriophage lambda.
restriction modification type ii endonucleases
Restriction/modification – Type II endonucleases

Frequencies of recognition sites:

4 bp: 44 = 256 nt

6 bp: 46 = 4096 nt

8 bp: 48 = 65536 nt (NotI cuts E. coli chromosome 21 times)

Product

Blunt end

Blunt end

5‘ overhang

3‘ overhang

Blunt end

5‘ overhang

5‘ overhang

5‘ overhang

slide22
Restriction recognitions sites can be unambiguous or ambiguous The enzyme BamHI recognizes the sequence GGATCC and no others - this is what is meant by unambiguous. In contrast, Hind II recognizes a 6 bp sequence starting with GT, ending in AC, and having a Pyrimidine at position 3 and a Purine at position 4
slide23
Most restriction enzymes bind to their recognition site as dimers (pairs), as depicted for the enzyme PvuII in the figure to the right.
mechanism of type ii restriction endonucleases
Mechanism of type II restriction endonucleases

Pingoud & Jeltsch (2001) Nucl. Acid Res. 29: 3705-3727.

patterns of dna cutting by restriction enzymes
Patterns of DNA Cutting by Restriction Enzymes
  • Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and phosphate groups. This leaves a phosphate group on the 5' ends and a hydroxyl on the 3' ends of both strands.
types of ends
Types of ends
  • 5' overhangs:
slide29
Different restriction enzymes can have the same recognition site - such enzymes are called isoschizomers
  • In some cases isoschizomers cut identically within their recognition site, but sometimes they do not
  • Sma I CCC GGG
  • Xma I C CCGGG
compatible cohesive ends
Compatible cohesive ends
  • Bam HI G↓GATCC
  • Bgl II A↓GATCT

GTG↓GATCCGT

CACCTAC↑CCA

GTG GATCCGT

CACCTAC CCA

CCA GATCTAA

GGTCTAG ATT

CCA↓GATCTAA

GGTCTAG↑ATT

GTGGATCTAA

CACCTACATT

setting up a digest
Setting up a digest
  • DNA: free from contaminants such as phenol or ethanol. Excessive salt will also interfere with digestion by many enzymes, although some are more tolerant of that problem.
  • An appropriate buffer: Different enzymes cut optimally in different buffer systems, due to differing preferences for ionic strength and major cation. When you purchase an enzyme, the company almost invariably sends along the matching buffer as a 10X concentrate.
  • The restriction enzyme! Remember these are generally expensive and heat labile
reaction conditions
Reaction conditions
  • 1. A double-stranded DNA sequence containing the recognition sequence.2. Suitable conditions for digestion.For example, BamHI has the recognition sequence: GGATCC and requires conditions similar to this:
  • 10 mM Tris-Cl (pH 8.0)5 mM Magnesium chloride100 mM NaCl1 mM 2-mercaptoethanolReaction conditions: 37 C
slide34
On the other hand, the enzyme Sma I has the recognition sequence: CCCGGG and requires conditions such as:
  • 33 mM Tris-acetate (pH 7.9)10 mM Magnesium acetate66 mM Potassium acetate0.5 mM DithiothreitolReaction conditions: 25 C
  • Most restriction enzymes are used at 37 C, however Sma I is an exception. Other examples of temperature exceptions are Apa I (30 C), Bcl I (50 C), BstEII (60 C), and Taq I (65 C). Taq I, by the way, is a restriction enzyme from the same type of organism that produces Taq polymerase (Thermophilus aquaticus, or Thermus aquaticus). Restriction enzyme names are based on a species-of-origin.
factors that influence restriction enzyme activity
Factors that Influence Restriction Enzyme Activity
  • Buffer Composition
  • Incubation Temperature
  • Influence of DNA Methylation
  • Star activity
slide36
Incubation Temperature The recommended incubation temperature for most restriction enzymes is 37°C. Restriction enzymes isolated from thermophilic bacteria require higher incubation temperatures ranging from 50°C to 65°C
methylase
Methylase
  • Dam methylase adds a methyl group to the adenine in the sequence GATC, yielding a sequence symbolized as GmATC.
  • Dcm methylase methylates the internal cytosine in CC(A/T)GG, generating the sequence CmC(A/T)GG.
slide38
The practical importance of this phenomenon is that a number of restriction endonucleases will not cleave methylated DNA.
slide39
The recognition site for ClaI is ATCGAT, which is not a substrate for Dam methylase. However , if that sequence is followed by a C or preceeded by a G, a Dam recognition site is generated and cleavage by ClaI is inhibited. Thus, a random sequence of DNA propagated in most strains of E. coli, half of the ClaI recognition sites will not cut.
star activity
Star Activity
  • When DNA is digested with certain restriction enzymes under non-standard conditions , cleavage can occur at sites different from the normal recognition sequence - such aberrant cutting is called "star activity". An example of an enzyme that can exhibit star activity is EcoRI; in this case, cleavage can occur within a number of sequences that differ from the canonical GAATTC by a single base substitutions
what causes star activity
What causes star activity
  • High pH (>8.0) or low ionic strength (e.g. if you forget to add the buffer)
  • Glycerol concentrations > 5% (enzymes are usually sold as concentrates in 50% glycerol)
  • Extremely high concentration of enzyme (>100 U/ug of DNA)
  • Presence of organic solvents in the reaction (e.g. ethanol, DMSO)
unit definition
Unit definition
  • The amount of enzyme needed to fully digest 1 ug of DNA in 1 hour
restriction enzymes cut an organism s dna into a reproducible set of restriction fragments
Restriction enzymes cut an organism’s DNA into a reproducible set of restriction fragments

Figure 7-6

slide45

3000 bp or 3K

Kpn I

Bam HI

Kpn I

Original plasmid

3500 bp

4k

Bam HI

2k

Digest of original plasmid

1k

slide49
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
slide50
When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.
  • Difference between DNA/RNA and proteins
slide51
Proteins and nucleic acids are electrophoresed within a matrix or "gel".

-agarose or polyacrylamide

agarose
Agarose
  • polysaccharide extracted from seaweed. It is typically used at concentrations of 0.6 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel.
  • non-toxic.
polyacrylamide
Polyacrylamide
  • is a cross-linked polymer of acrylamide. 3.5 and 20%.
  • Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates.
  • Acrylamide is a potent neurotoxin and should be handled with care
slide54
Uses
  • Polyacrylamide gels have a rather small range of separation, but very high resolving power. polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing in length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.
  • Agarose is used to separate DNA fragments from about 60 bp upward to 100,000 or so bp.
visualization of dna agarose
Visualization of DNA(Agarose)
  • Ethidium bromide, a fluorescent dye used for staining nucleic acids.
  • teratogen and suspected carcinogen and should be handled carefully.
  • Transilluminator (an ultraviolet light box)
slide57
Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.
slide59
Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass.
slide63
Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength.