2 Restriction Enzymes scan the DNA sequence Find a very specific set of nucleotidesMake a specific cut
3 Palindromes in DNA sequences Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands.5’3’3’5’
4 Restriction enzymes recognize and make a cut within specific palindromic sequences, known as restriction sites, in the DNA. This is usually a 4- or 6 base pair sequence.
5 Restriction Endonuclease Types Type I- multi-subunit, both endonuclease and methylase activities, cleave at random up to 1000 bp from recognition sequenceType II- most single subunit, cleave DNA within recognition sequenceType III- multi-subunit, endonuclease and methylase about 25 bp from recognition sequence
6 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ Hae IIIHaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases.5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’
7 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ Once the recognition site is found Hae III will cleave the DNA at that site5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’
8 These cuts produce “blunt ends” 5’ TGACGGGTTCGAGG CCAG 3’3’ ACTGCCCAAGGTCC GGTC 5’
9 The names for restriction enzymes come from: the type of bacteria in which the enzyme is foundthe order in which the restriction enzyme was identified and isolated.EcoRI for exampleR strain of E.coli bacteriaI as it is was the first E. coli restriction enzyme to be discovered.
10 “blunt ends” and “sticky ends” Hae III produced a “blunt end”? EcoRI makes a staggered cut and produces a “sticky end”5’ GAATTC 3’3’ CTTAAG 5’5’ GAATTC 3’3’ CTTAAG 5’5’ G AATTC 3’3’ CTTAA G 5’
15 Gene CloningWhat is gene cloning? How does it differ from cloning an entire organism?Why is gene cloning done?How is gene cloning accomplished ?What is a DNA ‘Library’?
16 What is DNA cloning?When DNA is extracted from an organism, all its genes are obtainedIn gene (DNA) cloning a particular gene is copied (cloned)
17 Why Clone DNA?A particular gene can be isolated and its nucleotide sequence determinedControl sequences of DNA can be identified & analyzedProtein/enzyme/RNA function can be investigatedMutations can be identified, e.g. gene defects related to specific diseasesOrganisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
18 How is DNA cloned?, I DNA is extracted- here from blood Blood sampleDNA is extracted- here from bloodRestriction enzymes, e.g. EcoR I, Hind III, etc., cut the DNA into small piecesDifferent DNA pieces cut with the same enzyme can join, or recombine.DNARestriction enzymes
19 The action of a restriction enzyme, EcoR I Note: EcoR I gives a ‘sticky’ end
20 DNA Cloning, IIBacterial plasmids (small circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzymeA chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant”
21 DNA cloning, IIIThe recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmidsThis insertion is called transformation
22 DNA Cloning, IVThe plasmids have naturally occurring genes for antibiotic resistanceBacteria containing plasmids with these genes will grow on a medium containing the antibiotic- the others die, so only transformed bacteria survive
23 DNA Cloning, VThe transformed bacterial cells form colonies on the mediumEach cell in a given colony has the same plasmid (& the same DNA)Cells in different colonies have different plasmids (& different DNA fragments)
24 Screening, I Screening can involve: Phenotypic screening- the protein encoded by the gene changes the color of the colonyUsing antibodies that recognize the protein produced by a particular gene
25 Screening, II3. Detecting the DNA sequence of a cloned gene with a probe (DNA hybridization)
27 PCR invented by Karry B. Mullis (1983, Nobel Prize 1993) patent sold by Cetus corp. to La Roche for $300 milliondepends on thermo-resistant DNA polymerase (e.g. Taq polymerase) and a thermal cycler
28 Heat-stable DNA polymerase Taq DNA polymerase was isolated from the bacterium Thermus aquaticus.Taq polymerase is stable at the high temperatures (~95oC) used for denaturing DNA.Hot springs at YellowstoneNational Park, Wyoming.
29 DNA polymerase requirements templateprimernucleotidesregulated pH, salt concentration, cofactors
30 Steps in DNA replication template denaturedprimers annealnew strand elongation
31 Steps in a PCR cycle 1) template denatured: 94 C, 30 sec 2) primers anneal45-72 C, depending on primer sequence30 sec – 1 min3) new strand elongation72 C depending on the type of polymerase1 min for 1000 nucleotides of amplified sequenceNumber of specific DNA molecule copies grows exponentially with each PCR cycle. Usually run cycles to get enough DNA for most applications (If you start with 2 molecules, after 30 cycles you will have more than a billion)
32 PCR Process 25-30 cycles 2 minute cycles DNA thermal cycler -For most cases the PCR process will undergo about cycles which will produce about 1 million copies of DNA that a scientist can perform other tests on.-Since each cycle is about 2 minutes long, it takes about an hour to reproduce 1 million copies of DNA.-The whole process of PCR takes place in a DNA thermal cycler which is pictured here.
34 Uses for PCR Research Clinical Gene cloning DNA fingerprinting Real-time PCRDNA sequencingClinicalDNA fingerprintingCrime scene analysisPaternity testingArcheological findsGenetically inherited diseases
36 Chain termination method (Sanger Method), sequence of single stranded DNA is determined by enzymatic synthesis of complementary strands which terminate at specific nucleotide positionsChemical degradation method (Maxam-Gilbert Method), sequence of a double stranded DNA molecule is determined by chemical treatment that cuts at specific nucleotide positions