DNA modifying enzymesEnzymes that modify DNA are useful because they allow the investigator to manipulate DNA in defined ways - Polymerases elongate DNA molecules by adding free nucleotides to the 3 ends (usually according to an opposite template strand) - Endonucleases cut DNA fragments in the middle of the molecule - Exonucleases degrade DNA from the ends - Ligases join loose ends of DNA together
DNA polymerasesDNA polymerases exonuclease activities: - Activity 35 exonuclease. ( proofreading activity) allows the polymerase to correct errors by removing a nucleotide that has been inserted incorrectly. - Activity 53 exonuclease activity is possessed by some DNA polymerases.
Proof reading activity of the 3 to 5 exonuclease. DNAPI stalls if the incorrect ntd is added - it cant add the next ntd in the chain
Proof reading activity is slow compared to polymerizing activity, but the stalling of DNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base.
The types of DNA polymerases used in research: DNA polymerase I: Unmodified E. coli enzyme . Use: DNA labeling . Klenow polymerase: Modified version of E.coli DNA polymerase I Use: DNA labeling
The Enzymologyof DNA Replication
In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I DNA Polymerase I has THREE different enzymatic activities in a single polypeptide: a 5 to 3 DNA polymerizing activity a 3 to 5 exonuclease activity a 5 to 3 exonuclease activity
Functional domains in the Klenow Fragment (left) and DNA Polymerase I (right).
DNA polymerase I
Figure 1. Prepare single-stranded template with Lambda Exonuclease.
Figure. 1 Lambda Exonuclease selectively digests the strand of a PCR product produced using a PCR primer with a 5-phosphate. The resulting singlestranded PCR product can be used for SSCP analysis or sequencing.
EndonucleasesI. Non specifice.g. S1 nuclease, from the fungus Aspergillus oryzae And Deoxyribonuclease I (DNaseI), from Escherichia coli
II. Specifice.g. Restriction endonucleases, from many sources
EndonucleasesI. Non specific - S1 nuclease (Endonuclease specific for singlestranded DNA and RNA, from the fungus Aspergillus oryzae Use:Transcript mapping - Deoxyribonuclease I (DNaseI) Endonuclease specific for double stranded DNA and RNA, from Escherichia coli Use:Nuclease footprinting
S1 nuclease protection digests only single-stranded RNA and DNA Find introns: intron
exon 2 genomic DNA antisense probe exon 1 exon 2
Digest with S1 Run gel
EndonucleasesII. Specific e.g. Restriction endonucleases: Sequencespecific DNA endonucleases, from many sources Use:Many applications
Restriction endonucleaseRestriction Endonucleases - Also called restriction enzymes - Recognize, bind to, and cleave DNA molecules at specific sequences (usually 4-6 base pairs in length) but there are some that are 5, 8, or longer - The double strand breaks can create ends that are: * Blunt, cutting both strands in the same place * Sticky, with overhanging nucleotides on the 5 or 3 ends
Restriction endonucleaseRestriction enzymes Over 10,000 bacteria species have been screened for restriction enzymes Over 2,500 restriction enzymes have been found Over 250 distinct specificities Occasionally enzymes with novel DNA sequence specificities are still found while most now prove to be duplicates (isoschizomers) of already discovered specificities.
Restriction endonucleaseThere are three types of restriction enzymes. With Types I and III there is no strict control over the position of the cut relative to the specific sequence in the DNA molecule that is recognized by the enzyme. These enzymes are therefore less useful . Type II enzymes do not suffer from this disadvantage because the cut is always at the same place, either within the recognition sequence or very close to it
Type II Restriction enzymes are endonucleases that cut DNA at specific sites, and are most useful for molecular biology research
Restriction enzymes are molecular scissors
Restriction Enzymes scan the DNA code Find a very specific set of nucleotides Make a specific cut
Restriction enzymesRestriction enzymes recognize and make a cut within specific palindromic sequences, known as restriction sites, in the genetic code. This is usually a 4- or 6 base pair sequence.
Picking a palindromeWords that read the same forwards as backwards hannaH Hannah Level Madam leveL madaM
Restriction enzymesRestriction Enzyme Recognition Sites Restriction sites are general palindromic: Able was I, ere, I saw Elba
5-GGATCC-3 Bam H1 site: 3-CCTAGG-5
HaeIIIHaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases.
5 TGACGGGTTCGAGGCCAG 3 3 ACTGCCCAAGGTCCGGTC 5 5 TGACGGGTTCGAGGCCAG 3 3 ACTGCCCAAGGTCCGGTC 5
Once the recognition site was found HaeIII could go to work cutting (cleaving) the DNA 5 TGACGGGTTCGAGGCCAG 3 3 ACTGCCCAAGGTCCGGTC 5
These cuts produce what scientists call blunt ends5 TGACGGGTTCGAGG 3 ACTGCCCAAGGTCC CCAG 3 GGTC 5
Restriction enzymesRestriction enzymes are named based on the bacteria in which they are isolated in the following example for the enzyme EcoRI: E Escherichia (genus) co coli (species) R RY13 (strain) I First identified Order ID'd in bacterium
Restriction enzymesNomenclature of Restriction Enzymes The 1st letter (in capital and italics) = first initial of Genus name (from which the enzyme was isolated The 2nd and 3rd (in italics) = the first 2 letters of the species name e.g. Hin = Haemophilus influenzae The 4th letter (sometimes in italics) = strain or type e.g. Hind = Haemophilus influenzae Rd The roman number followed is given to distinguish different restriction and modification system in the same strain e.g. HindIII
EcoRI 5 G AATTC 3 3 CTTAA G 5
W=A or T
blunt ends and sticky endsRemember how HaeIII produced a blunt end? EcoRI, for instance, makes a staggered cut and produces a sticky end 5 GAATTC 3 3 CTTAAG 5 5 GAATTC 3 3 CTTAAG 5 5 G 3 CTTAA AATTC 3 G 5
Restriction enzymesSingle stranded nick
Eco RI Restriction Enzyme
Some more examples of restriction sites of restriction enzymes with their cut sites: HindIII: 5 AAGCTT 3 3 TTCGAA 5 BamHI: 5 GGATCC 3 3 CCTAGG 5 AluI: 5 AGCT 3 3 TCGA 5
Restriction Enzyme Recognition SitesBglII 5 A-G-A-T-C-T T-C-T-A-G-A 5
5 G-A-T-C C-T-A-G 5
All these sticky ends are compatible
5 G-G-A-T-C-C C-C-T-A-G-G 5
Isoschizomers: In certain cases, two or more different enzymes may recognize identical sites. (e.g. MboI also cleaves at GATC, and so is an isochizomer of Sau3A.)
Restriction enzymesFrequency of cutting of recognition enzymes Sau 3A (GATC) cuts ()()()() = once every 256 base pairs (assuming G/C = A/T, which is often does not) BamH1 (GGATCC) cuts ()()()()()() = once every ~4Kb HindII (GTPyPuAC) cuts ()()()()()() = once every ~1Kbhttp://tools.neb.com/NEBcutter2/index.php
Ligation of compatible sticky endsHuman DNA cleaved with EcoRI 5-C-G-G-T-A-C-T-A-G-OH 3-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4 Corn DNA cleaved with EcoRI
Complementary base pairing5-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3 3-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5
+ DNA Ligase, + rATP
recombinant DNA molecule
Exercise1HindIII 1/ 6160
EcoRI 5660 PvuII 5116
YIP MApal 2035 PvuII 3547 SmaI 2860 SmaI 5 ccc ggg 3 How many base pairs in this plasmid? How mamy fragments will be produced if this plasmid is digested with PvuII?
Agarose Gel Electrophoresis
_DNA is negatively charged from the phosphate backbone Agarose mesh
Visualize DNA with ethidium bromide fluoresces orange ONLY when bound to DNA
Gel Electrophoresis of DNA
What is Gel Electrophoresis? Electro = flow of electricity, phoresis, from the Greek = to carry across A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied Charged particles can include DNA, amino acids, peptides, etc
Gel electrophoresisGel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.
Why do gel electrophoresis? When DNA is cut by restriction enzymes, the result is a mix of pieces of DNA of different lengths It is useful to be able to separate the pieces - i.e. for recovering particular pieces of DNA, for forensic work or for sequencing
Gel with molecular weight marker
Summary Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands This allows very specific cutting of DNAs 4-7 The cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to form a recombinant DNA in vitro