Molecular Genetics 7 777120 Lab 1 Objectives Define restriction enzymes and their role in genetic engineering Understand the

he principles and applications of restriction enzymes and restriction digestion of DNA Become proficient in the loading of agarose gels Introduction Lambda () is a virus that infects bacteria. his virus does not infect humans and is! therefore! safe for us to study. "n this investigation! you #ill observe the effects of three restriction enzymes on DNA. A restriction enzyme (also $no#n as an endonuclease) is an enzyme that recognises a specific base pair se%uence #ithin DNA and cuts the DNA at that se%uence. he enzyme #ill cut every time the se%uence it recognises appears #ithin the DNA molecule. he DNA fragments generated are called restriction fragments. hese fragments can be separated from each other according to their size by gel electrophoresis. he restriction enzymes you #ill use are Eco&"! Pst" and Hind""". Ne't #ee$! you #ill use gel electrophoresis to separate the resulting DNA pieces. "nformation in your te'tboo$( Lambda () DNA &estriction enzymes( &estriction mapping( /el electrophoresis( pp )*+** pp ,-+,* pp ,*+,. pp 00+01

Part A: Agarose Gel Electrop oresis Materials #o large 23 agarose4 5.16 B7 gels 5.16 B7 electrophoresis buffer! -L per large gel tan$ for running gel ris47D A buffer ( 7) p8 ..5! 05 microfuge tubes ' 255 L 9ini agarose gel electrophoresis trays 9ini agarose gel electrophoresis combs 9ini agarose gel electrophoresis tan$s :o#er supplies ;terile -15ml flas$s Agarose po#der <eigh boats Balance 5.16 B7 electrophoresis buffer! )5 ml per small gel tray for ma$ing gel. 5.16 B7 electrophoresis buffer! -15 ml per small gel tan$ for running gel ;terile255ml measuring cylinders ;terile-15mL measuring cylinders

7thidium bromide (25 mg4mL) ;ample loading buffer! 05 microfuge tubes ' 255 L 9icrofuge test tubes! 2.1 l 9icrofuge test tube trays ;afety glasses Met od A!1 "#o be done individuall$% =ou have been provided #ith t#o 23 agarose gels already made up. =ou are to use these gels to practice loading samples. =ou #ill need to ta$e turns #ith others in the class. 2. a$e three 2.1 mL and place into a microfuge test tube tray.

2. :ipette 1L of sample loading buffer into each tube. 3. :ipette 21 L of 7 buffer into each tube. ,. 7nsure the tube lids are closed properly and flic$ tubes to mi' contents. 1. Briefly spin tubes (1 sec) in microfuge (ensure tubes are balanced) to bring contents to the bottom of the tube. 6. :ipette 2. L of 7 buffer into each of three #ells on one of the gels provided. =our lecturer #ill demonstrate ho#. Met od A!2 "&or' in groups o( t ree% =ou #ill learn ho# to pour an agarose gel. CAUTION: WEAR GLOVES 2. <eigh out )g of agarose po#der into a #eigh boat and transfer to a sterile -15ml flas$. -. :our in )5 ml 5.16 B7 electrophoresis buffer. 0. 9elt agarose in micro#ave > you lecturer #ill sho# you ho#. CAUTION: Do not allow agarose solution to boil over I! "ou #a$e a #ess in t%e #i&rowave "ou #ust &lean it u' "oursel! be!ore "ou &an 'ro&ee( ,. Allo# the agarose to cool do#n to a temperature at #hich you can hold the flas$ reasonably comfortably in your hands. 1. Assemble gel rig4tray and comb ready for the agarose to be poured in. 6. Add 2 L ethidium bromide to cooled agarose? s#irl gently to mi'. )A*#IO+: Et%i(iu# bro#i(e is a #utagen Ensure "ou are wearing gloves an( sa!et" glasses w%en (is'ensing et%i(iu# bro#i(e an( 'ouring "our gel *. :our into assembled gel rig4tray in one movement? remove any air bubble as sho#n by your lecturer. .. Leave gel to set. )A*#IO+: To avoi( &reating bubbles or ri''les) (o not tou&% gel rig*tra" w%ile gel is setting @. Ance gel has set! remove comb and pour -15mL 5.16 B7 into gel rig.

25. "f there is time! repeat method A.2 using your o#n gel and t#o microfuge tubes. =ou #ill have . #ells to load samples into. 7ach of the three in your group #ill have t#o #ells into #hich you can practice loading a sample.

Part ,: -estriction .igest Materials Hind""" restriction enzyme (25 units4 L) B&oche Applied ;ciences C 10656313001]( 25 microfuge tubes ' 1L Pst" restriction enzyme (25 units4 L) B&oche Applied ;ciences C 10621625001]( 25 microfuge tubes ' 1L Eco&" restriction enzyme (25 units4 L) B&oche Applied ;ciences C 10703737001]( 25 microfuge tubes ' 1L 256 &estriction buffer 8 B&oche Applied ;ciencesD( 25 microfuge tubes ' -5uL 256 &estriction buffer B B&oche Applied ;ciencesD( 25 microfuge tubes ' -5uL Lambda DNA! uncut B&oche Applied ;ciences C 10745782001]: (-15 ng4L) 9icrofuge test tubes! 2.1 ml 9icrofuge test tube trays AdEustable micropipettes! 5+- L AdEustable micropipettes! ->-5 L AdEustable micropipettes! -5>-55 L ;terile pipette tips for each type of adEustable micropipette 9icro#ave oven 9icrofuges Met od ,!1: "&or' in groups o( t ree% =ou are to set up si' restriction digests of (Lambda) DNA. 7ach digest #ill be set up in a separate tube. 2. a$e si' 2.1 ml microfuge tubes and label each tube #ith your name and each tube as FAG! FBG FHG! FDG! F7G and FIG. 8old your tubes in a microfuge tray. -. ;et up the restriction enzyme reactions by adding each of the ingredients listed belo# for each tube. Add the reaction ingredients in the order listed.

#ube

.+A "2/0ng0 L% ,L ,L ,L ,L ,L ,L

1terile 102 102 deionised ,u((er ,u((er &ater 3 , 20L 20L 20L 2-L 2-L 2-L -L -L + -L -L -L + + -L + + +

E&o-I "10*0 L% 2L + + 2L 2L +

+stI "10*0 L% + 2L + 2L + 2L

,indIII "10*0 L% + + 2L + 2L 2L

A B H D 7 I

0. 7nsure the tube lids are closed properly and flic$ tubes to mi' contents. ,. Briefly spin tubes (1 sec) in microfuge (ensure tubes are balanced) to bring contents to the bottom of the tube. 1. "ncubate overnight in 0*oH #arm room.

-estriction En4$5es
1e6uence recognition 7 cleavage: he names of the enzymes are derived from the bacterium from #hich they #ere isolated. e.g. Eco&" is purified from Escherichia coli. Bam8" is purified from a Bacillus amyloliqueraciens. "n some cases! more than one restriction enzyme can be purified from the same organism. "n this case! the enzyme names have different numerals after the bacteriumGs name. e.g. Pvu" and Pvu"" are different restriction enzymes from the same organism! Proteus vulgaris. he most useful restriction enzymes recognise ,+. base pair sites! of symmetric inverted repeats! called palindromes. "n the e'ample! Eco&"! notice ho# the 1G to 0G se%uence on top J bottom strands is identical( Eco&" 1G+/AA H+0G 0G+H AA/+1G

;ome restriction enzymes cut both DNA strands at the same position! creating blunt+ ended fragments. 8o#ever! the maEority of enzymes ma$e cuts staggered on each strand! resulting in a fe# base pairs of single+stranded DNA at each end of the fragment! $no#n as Fstic$yG ends. hese #ill either be 1G overhangs or 0G overhangs. Hleavage results in either a 1G overhang! 0G overhang or a blunt end. o Hommonly used enzymes that give a 1G overhang ( Eco&"! Bam8"! Xba"! Hind""" o Hommonly used enzymes that give a 0G overhang( Pst"! Kpn"! Pvu"! Sac" o Hommonly used enzymes that give a blunt end( Sma"! Eco&K &estriction enzymes #ith shorter recognition se%uences cut more fre%uently than those #ith longer recognition se%uences. Ior e'ample! a , base pair (bp) cutter #ill cleave! on average ,, (-1)) bases! #hile a ) bp cutter cleaves every ,) (,5@)) bases. ;ome enzymes have degenerate recognition se%uences! i.e. they can recognise and cleave several similar but different sites. Ior e'ample( o Ava"" cuts at //AHH as #ell as // HH. o Ava" cuts at H:yH/:u/. :y stands for pyrimidine i.e. or H! #hile :u stands for purine i.e. A or /. hus! Ava" #ill digest H H/A/! H H///! HHH/A/ and HHH///. o Dde" cuts at H NA/! N being any bases i.e. A! H! / or . hus! it cleaves H AA/! H HA/! H /A/! H A/ "soschizomers are different enzymes that share the same specificity! i.e. recognise the same site. hese are derived from different bacteria. o e.g. Sau0A is an isoschizomer of Mbo" > they both cleave the site /A H. 8o#ever! in this e'ample Mbo" does not cleave methylated DNA! #hile Sau0A does. Sau0A can therefore be used instead of Mbo" #here necessary. ;uch differences can be very useful. o Ather isoschizomers recognise a subset of another enzymeGs site. Ior e'ample! Sau0A (/A H) #ill recognise! cleave and leave the same overhang as Bam8" (//A HH) and Bgl"" (A/A H ).

he type of restriction digest done depends on the do#nstream usage of the DNA fragments. Ior e'ample! DNA is digested #ith restriction enzymes a) for restriction mapping DNA molecules and b) prior to cloning. -estriction 5apping "t is possible to determine the relative positions of a variety of restriction sites in a fragment of DNA #ithout se%uencing it. his process is called restriction mapping. o ensure the greatest accuracy! a large panel of restriction enzymes are generally used to digest the DNA fragment of interest. After determining #hich enzymes actually cut! the restriction sites #ould then be ordered to generate a linear map. his is largely a trial+and+error process! so the more enzymes used! the more accurate the restriction map #ill be. An e'ample restriction map for the plasmid pB&0-- is given belo#(

Iigure 2( &estriction map of the plasmid! pB&0-- (2). )loning &estriction enzymes are essential tools for cloning fragments of DNA. 8o# they are used for this purpose #l be discussed in more detail in a later lecture J lab class.

# e -estriction .igest -eaction he components of a restriction digest reaction are( #ater? DNA? buffer? restriction enzyme. his seems straightfor#ard but there are several things to consider. &estriction enzymes have specific re%uirements to #or$ at their optimum. he salt concentration! the p8! the temperature! divalent cations! reducing reagent and carrier protein are most of the parameters that must be considered. 8ater 9ust be free of contaminants! especially DNAse #hich can brea$ do#n the DNA. herefore! sterile! distilled #ater is used. .+A <hat is the do#nstream purpose of the digestL "s it for cloningL "s it for :H&L "s it for something elseL he amount of DNA digested depends on the do#nstream application( for mapping of cloned DNA! 5.-+2g DNA per reaction is enough! #hile for ;outhern blotting of mammalian genomic DNA! a minimum of 25g DNA per reaction is recommended. ,u((er he buffer provides the optimal p8! salt concentration! as #ell as other components that may be re%uired. &estriction enzymes #or$ in either no salt! lo# salt! medium salt or high salt concentrations. ;ome have e%ual activity under more than one of these conditions. his can be useful #hen DNA is to be digested by t#o restriction enzymes? for them to go into the same reaction at the same time! their buffer re%uirements must be compatible. 9ost companies test their enzymes under a variety of conditions and determine the optimal conditions for digestion for their enzyme. hese days! commercially bought enzymes are generally delivered #ith a buffer that is optimal. 9any enzymes #ill #or$ #ell in the same buffer so that digests #ith more than one enzyme can be possible. Bufffers are provided as concentrated stoc$s! generally as 16 or 256 stoc$s. hey must be diluted to 26 in the digest for the restriction enzymes to #or$. #e5perature he optimal temperature is generally a reflection of the organism from #hich the enzyme #as derived. Ior e'ample( Eco&" comes from Escherichia coli! #hich has an optimal gro#ing temperature of 0* oH. he temperature for optimal activity for Eco&" is also 0* oH. aq" comes from the deep+sea vent organism hermus aquaticus. "ts optimal gro#ing temperature is )1 oH. "f t#o restriction enzymes are to be used in the same reaction at the same time! their temperature optima must be compatible. -estriction en4$5e he things to consider #ith these enzymes include #hich enzyme should be used! ho# much of it needs to be used and for ho# longL <hich enzyme(s) you use depends on #hat sites are available #ithin the DNA you are cutting and #hat the do#nstream use of the digested DNA #ill be. Ior e'ample! if you #ish to clone a piece of DNA into a plasmid vector! you need to choose an enzyme(s) #hich does not cut #ithin

the fragment you #ish to clone but is also compatible #ith the cloning site #ithin the target vector you #ish to use. 7ach restriction enzyme is supplied as a given number of units! corresponding to a specified level of enzyme activity. 7ach supplier ships their enzyme #ith a data card that indicates the units of activity! the concentration of enzyme! optimal reaction conditions! storage conditions! contamination! etc. 8istorically! one unit (2U) of an enzyme (i.e. the specific activity) #as defined as the amount of enzyme re%uired to digest 2g Lambda DNA in 2 hour at its optimal temperature. his is no# outdated since many enzymes donGt cut Lambda. Ior e'ample! the company Ne# 7ngland Biolabs defines a unit as MAne unit of restriction endonuclease activity is defined as the amount of enzyme re%uired to completely digest 2 g of substrate DNA in a total reaction volume of 5.51 ml in one hour using the N7B buffer provided.N his means that 2Unit of enzyme should cut AN= DNA under the conditions defined. /enerally! ho#ever! for more comple' DNA molecules such as genomic DNA or supercoiled plasmid DNA! more enzyme is re%uired for longer than for simple molecules such as rela'ed plasmids. 9ost researchers add a ten+fold e'cess of enzyme to their reactions in order to ensure complete cleavage. &estriction enzymes are supplied by the companies in glycerol. his chemical prevents the proteinaceous enzyme from freezing #hen stored at >-5 oH (the optimal storage temperature). "f an enzyme froze it #ould become inactive. 8o#ever! too much glycerol in a restriction digest can interfere #ith the enzymeGs activity. hus! the rule of thumb is not to have more than 253 of the total reaction volume as restriction enzyme -eaction volu5e 9ost digests are carried out in a volume bet#een 25 and 15 OL. &eaction volumes smaller than25 OL are susceptible to pipetting errors! and are not recommended. # e botto5 line is9999,e(ore setting up a restriction digest: c ec' to 5a'e sure t at $ou are using t e proper conditions;

-e(erences
2. http(44d#b.unl.edu4 eacher4N;I4H5.4H5.Lin$s4###.fermentas.com4 ech"nfo4NucleicAc ids4mappbr0--.htm do#nloaded 20th Puly! -551. -. http(44###.roche+applied+science.com4pac$+insert45*50*0*a.pdf . "nformation regarding Eco&"! do#nloaded 22th Puly! -551 0. http(44###.roche+applied+science.com4pac$+insert45)1)020a.pdf . "nformation regarding Hind"""! do#nloaded 22th Puly! -551. 4. http(44###.roche+applied+science.com4pac$+insert45)-2)-1a.pdf . "nformation regarding Pst"! do#nloaded 22th Puly! -551.