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Lab. Restriction Enzymes Objectives: 1- Introduce the students to digest genomic DNA by restriction endonucleases 2- Observe the results of digestion on agarose gel electrophoresis. Introduction: Restriction enzymes (also known as restriction endonucleases) found in bacteria (and harvested from them for use) and cut DNA up at specific sequences in the genome. For example, the commonly used restriction endonuclease EcoRI recognizes every point in DNA with the sequence GAATTC, and cuts at the point between the Guanine and Adenine. Interestingly, the recognition sequences for most restriction endonucleases are genetic palindromic, e.g., the sequence reads exactly the same backwards on the complementary strand. Examples of restriction enzymes include: Enzyme Source Recognition Sequence Cut 5'GAATTC 3'CTTAAG 5'---G AATTC---3' 3'---CTTAA G---5' 5'CCWGG 3'GGWCC 5'--CCWGG---3' 3'---GGWCC ---5' BamHI Bacillus amyloliquefaciens 3'CCTAGG 5'GGATCC 5'---G GATCC---3' 3'---CCTAG G---5' HindIII Haemophilus influenzae 5'AAGCTT 3'TTCGAA 5'---A AGCTT---3' 3'---TTCGA A---5' TaqI Thermus aquaticus 5'TCGA 3'AGCT 5'---T CGA---3' 3'---AGC T---5' HinfI Haemophilus influenzae 5'GANTC 3'CTNAG 5'---G ANTC---3' 3'---CTNA G---5' Sau3A Staphylococcus aureus 5'GATC 3'CTAG 5'--GATC---3' 3'---CTAG ---5' PovII* Proteus vulgaris 5'CAGCTG 3'GTCGAC 5'---CAG 3'---GTC CTG---3' GAC---5' SmaI* Serratia marcescens 5'CCCGGG 3'GGGCCC 5'---CCC 3'---GGG GGG---3' CCC---5' HaeIII* Haemophilus aegyptius 5'GGCC 3'CCGG 5'---GG 3'---CC EcoRV* Escherichia coli 5'GATATC 3'CTATAG 5'---GAT 3'---CTA EcoRI Escherichia coli EcoRII Escherichia coli * = blunt ends N = C or G or T or A W = A or T 1 CC---3' GG---5' ATC---3' TAG---5' Restriction enzymes are part of a bacteria's ''immune'' system. These are enzymes that cut DNA at specific sites (typically a four or a 6 base-pair sequence). Bacterial DNA is modified to be protected by methylation while foreign DNA, such as incoming viruses, are not. Usually, organisms that make restriction enzymes also make a companion modification enzyme ( DNA methyltransferase) that protects their own DNA from cleavage. These enzymes recognize the same DNA sequence as the restriction enzyme they accompany, but instead of cleaving the sequence, they disguise it by methylating one of the bases in each DNA strand. Thus, we can take a long piece of DNA and cut it with a restriction enzyme, generating numerous fragments. Even a single-base change will destroy a restriction enzyme target site. Likewise, even if a site is the same in two molecules, the lenght of DAN sequence between them may change. Thus if two DNA molecules differ in sequence, they likely have different lengths for the fragments produced following treatment with restriction enzymes. Typical digestions included a unit of enzyme per microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC. 2 The Digests: 1. Prepare the reaction for restriction digestion by adding the following reagents in the order listed to a microcentrifuge tube: Example:MTHFR PCR products Components of the reaction Volum PCR product 14 µl 2 µl 2 µl 2 µl 20 µl dH20 Restriction buffer 10x Hinf1 enzyme (1-10 units Total volume per ug DNA) 2. Gently mix by pipetting and incubate the reaction at the appropriate temperature (typically 37degC) for 1-3 hours or overnight. 3. Inactivate the enzyme(s) by heating at 70-100degC for 10 minutes. 4. Run on the agarose gel 2%, o freeze the samples until you have time to run. 5. Record your observation . 3