WSSP-14 Chapter 3 Analyzing DNA –Restriction Digests © 2014 WSSP

p Cloning cDNA fragments into pTripleEX2 © 2014 WSSP

Restriction enzymes are used for cloning and analyzing DNA fragments © 2014 WSSP

ONs 1234 MPs M1234 RI/HindIII M1234 PstI

ONs 1234 MPs M1234 RI/HindIII

Bacteriophage - "eaters of bacteria" © 2014 WSSP

Restriction enzymes - endonucleases, Cleave a specific DNA sequence Protect bacteria from phage infection, digest phage DNA after infection p. 3-1 GAATTC CTTAAG G CTTAA AATTC G © 2014 WSSP

Why don't the restriction enzymes degrade the cell DNA? © 2014 WSSP

Cellular DNA protected by methylases - block restriction enzyme activity p. 3-1 GAATTC CTTAAG G CTTAA AATTC G GAATTC CTTAAG M M Methylase GAATTC CTTAAG M M EcoRI © 2014 WSSP

DNA-binding proteins make non-covalent contacts to the DNA Methyl group CH 3 Methylation blocks binding by the protein

NOTE: DNA Methylation in bacteria has a different purpose then DNA methylation in eukaryotes Vertebrate CpG methylation – reduces gene expression in adult somatic cells, hypermethylation silences genes Genomic imprinting, X-chromosome inactivation, suppression of repetitive elements Bacterial Dam methylase (GATC) involved in mismatch repair, replication, and gene expression © 2014 WSSP

Each strain of bacteria has a specific set of restriction enzymes EcoRI from Escherichia coli BamHI from Bacillus amyloliqueraciens PvuI and PvuII are different enzymes from same strain. Originally purified by individual labs, Nathans, Smith Now supplied by companies - NEB, Promega p. 3-1 © 2014 WSSP

Sequence Recognition and cleavage: a) 5' overhangEcoRIGAATTCG OH p AATTC CTTAAGCTTAA p OH G b) 3' overhang KpnIGGTACCGGTAC OH p C CCATGGC p OH CATGG c) Blunt endSmaICCCGGGCCC OH p GGG GGGCCCGGG p OH CCC p. 3-2 © 2014 WSSP

DNA fragments with compatible sticky ends can be ligated together Example: NNG AATTCNNNNNGAATTCNNN NNCTTAA GNNNNNCTTAAGNNN Not NNGGGCCCNNNNNGGGCCCNNN NNCTTAA GNNNNNCTTAAGNNN Ligase +ATP OHP P P P © 2014 WSSP

DNA fragments with blunt ends can be ligated together Example: NNGAATTCNNNNNGAATTCNNN NNCTT AAGNNNNNCTTAAGNNN NNGAACCCNNNNNGAACCCNNN NNCTTGGGNNNNNCTTGGGNNN Ligase OHP P P P © 2014 WSSP

Sequence Recognition and cleavage: d) Degenerate: AvaII GGWCC:GGTCC, GGACC AvaI CPyCGPuG CTCGAG Py stands for pyrimidine- T or C CTCGGG Pu stands for purine - A or G CCCGAG CCCGGG CTCGAGCCCGGGCCCGAG GAGCTCGGGCCCGAGCCC DdeI CTNAG: CTAAG, CTGAG, CTCAG, CTTAG BbsI cleaves GAAGACNN CTTCTGNNNNNN p. 3-2 © 2014 WSSP

Recognition sites for the SfiI restriction enzyme p. 3-2 © 2014 WSSP

p. 3-3 © 2014 WSSP

Activity: in units which corresponds to a specified level of enzyme activity. NEB defines a unit as: “One unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1  g of substrate DNA in a total reaction volume of 0.05 ml in one hour using the NEB buffer provided.” p. 3-3 © 2014 WSSP

Before setting up a restriction digest check to make sure that you are using the proper conditions! Improper conditions can denature (inactivate) the enzyme or cause non-specific digestion (Star activity) Activity of an enzyme can change under different conditions: pH- 7.5, 8.0, 8.5 salt concentration- 20 mM, -150 mM divalent cations- Mg++ reducing reagent- DTT carrier protein-BSA temperature- 37C, RT, 60C Restriction enzymes are proteins with optimal conditions p. 3-4 © 2014 WSSP

p. 3-4 Information on Restriction Enzymes © 2014 WSSP

4. Set up the Reaction: Add in the following order: Sterile ddH 2 O7.0  l 10 X restriction buffer 2.0  l Miniprep DNA (0.5  g) 10.0  l Enzyme (20 U/  l)1.0  l Total volume20.0  l The two most important rules in enzymes Always keep enzymes on ice or in a cooler. Always use a fresh tip when pipeting from the enzyme stocks. p. 3-5 © 2014 WSSP

Set up a master mix multiple digests: SingleMultiple Sterile ddH 2 O7.0  l35.0  l 10 X restriction buffer 2.0  l10.0  l Miniprep DNA (0.5  g) 10.0  l**none** Enzyme (20 U/  l)1.0  l5.0  l Total volume20.0  l 10.0  l Mix aliquot 10.0  l DNA p. 3-7 Never put anything into your yellow MP tube!!! © 2014 WSSP

5. Incubate reactions at the appropriate temperature for the appropriate time. Usually 37˚C and incubate 1 hr or more. 6. If running on a gel: Add gel loading dye EDTA - Stops reaction. Dyes (BPB and XC) - to help see sample while loading and monitor electrophoresis Glycerol - so sample sits at bottom of the well p. 3-6 © 2014 WSSP

Using a restriction digest to map a plasmid p. 3-8 © 2014 WSSP

Map of the pTriplEX2 plasmid p. 3-9 cDNA Fragment © 2014 WSSP

Why not test cut the plasmid with SfiI? 2. Can’t tell if single cut or non insert 3. It is also expensive. p May have lost the site in cloning © 2014 WSSP

Theoretical PvuII-HF digests of plasmids p © 2014 WSSP

Why are there 3 bands in the uncut lane? p © 2014 WSSP

A real Digest gel of clones from the Duckweed cDNA library p © 2014 WSSP

5. Does your digest look complete? Are the intensities of the bands proportional to the size? p © 2014 WSSP

Bands Generated from Partial Digests p © 2014 WSSP

6. Is there a PvuII site in the insert? Are there more than two bands? p © 2014 WSSP

6. Is there a PvuII site in the insert? Are there multiple bands? p © 2014 WSSP

7. What is the size of the insert? p bp bp = 0 bp 2.9 Kb © 2014 WSSP

8. Do the results from the digest and the PCR agree? p bp bp = 0 bp 700 bp bp = 0 bp © 2014 WSSP

8. Do the results from the 20JM1.10 digest and the PCR agree? p bp bp = 400 bp 1100 bp bp = 400 bp © 2014 WSSP

8. Do the results from the 20JM2.10 digest and the PCR agree? p bp bp = 1500 bp 2200 bp bp = 1500 bp © 2014 WSSP

8. How do we figure out the insert size from the 20JM4.10 digest? p bp bp- 700 bp = 900 bp 2900 bp 1200 bp 400 bp © 2014 WSSP

8. Do the results from the 20JM4.10 digest and the PCR agree? p bp bp = 900 bp 1200 bp +400 bp- 700 bp = 900 bp

Enter the results of the RD gel and whether the clone should be sequenced into your school’s Google Docs Clone Report sheet © 2014 WSSP

Enter the conclusions into the Google Docs Clone report © 2014 WSSP