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Genetic Engineering

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

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  1. Genetic Engineering • technology involved in removing, modifying, or adding genes to a DNA molecule • Aka recombinant DNA technology

  2. Restriction endonucleases • Gel electrophoresis • Cloning vectors • Simple cloning exercise

  3. 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

  4. Restriction Endonucleases

  5. 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.

  6. All restriction enzymes cut DNA between the 3’ carbon and the phosphate moiety of the phosphodiester bond.

  7. 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

  8. Named after the organism from which they were derived • EcoRI from Escherichia coli • BamHI from Bacillus amyloliquefaciens

  9. Availability • Over 200 enzymes identified, many available commercially from biotechnology companies

  10. 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

  11. Restriction/modification systems - EcoRI restriction enzyme EcoRI methylase EcoRI Meth. EcoRI

  12. 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

  13. 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)

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  21. 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

  22. Most restriction enzymes bind to their recognition site as dimers (pairs), as depicted for the enzyme PvuII in the figure to the right.

  23. Mechanism of type II restriction endonucleases Pingoud & Jeltsch (2001) Nucl. Acid Res. 29: 3705-3727.

  24. 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.

  25. Types of ends • 5' overhangs:

  26. 3' overhangs

  27. Blunts

  28. 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

  29. Restriction fragments with complementary “sticky ends” are ligated easily

  30. 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

  31. 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

  32. 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

  33. 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.

  34. Factors that Influence Restriction Enzyme Activity • Buffer Composition • Incubation Temperature • Influence of DNA Methylation • Star activity

  35. 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

  36. 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.

  37. The practical importance of this phenomenon is that a number of restriction endonucleases will not cleave methylated DNA.

  38. 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.

  39. 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

  40. 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)

  41. Unit definition • The amount of enzyme needed to fully digest 1 ug of DNA in 1 hour

  42. Restriction enzymes cut an organism’s DNA into a reproducible set of restriction fragments Figure 7-6

  43. 3000 bp or 3K Kpn I Bam HI Kpn I Original plasmid 3500 bp 4k Bam HI 2k Digest of original plasmid 1k

  44. Electrophoresis

  45. Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.

  46. 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

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