DNA -- Teacher Preparation Notes

by Dr. Jennifer Doherty and Dr. Ingrid Waldron, Department of Biology, University of Pennsylvania, 20111

ea!hing Points D"# is a n$!lei! a!id made of t%o strands of n$!leotides %o$nd together in a spiral !alled a do$ble heli&. 'a!h n$!leotide is !omposed of a s$gar mole!$le (no%n as deo&yribose, a phosphate gro$p, and one of fo$r different nitrogeno$s bases) adenine *A+, thymine *T+, g$anine *G+, or !ytosine *C+. he phosphate and s$gar parts of the n$!leotides form the ba!(bone of ea!h strand in the D"# do$ble heli&. he bases e&tend to%ard the !enter of the do$ble heli&, and ea!h base in one strand is mat!hed %ith a !omplementary base in the other strand, in a!!ord %ith the base,pairing r$les) A pairs %ith T and G pairs %ith C. hese !hara!teristi!s are the same for the D"# of all organisms. he D"# of different organisms differs in the se-$en!e of n$!leotides, and these differen!es in n$!leotide se-$en!e are responsible for the geneti! differen!es bet%een different organisms. D"# repli!ation prod$!es t%o ne% D"# mole!$les that are identi!al to the original D"# mole!$le, so ea!h of the ne% D"# mole!$les !arries the same geneti! information as the original D"# mole!$le. D$ring D"# repli!ation, the t%o strands of the original D"# do$ble heli& are separated and ea!h old strand is $sed as a template to form a ne% D"# strand. he en.yme D"# polymerase adds n$!leotides one,at,a,time, $sing the base,pairing r$les to mat!h ea!h n$!leotide in the old D"# strand %ith a !omplementary n$!leotide in the ne% D"# strand. h$s, ea!h ne% D"# do$ble heli& !ontains one strand from the original D"# mole!$le, together %ith a ne%ly synthesi.ed mat!hing D"# strand. In e$(aryoti! !ells, ea!h !hromosome !onsists of D"# %rapped aro$nd proteins. he !hromosomes are !ontained in the n$!le$s inside a n$!lear membrane. Equipment and Supplies: /ports Drin( li(e 0atorade *10 m1 per st$dent+ 1i-$id dish soap *0.22 m1 per st$dent+ 3eat enderi.er *a pin!h per st$dent+ 40,526 isopropyl or ethyl al!ohol *7 m1 per st$dent+ /tring for ne!(la!e *2.2 ft per st$dent+ 8 o.. Di&ie !$ps *1 per st$dent+ $b of i!e, free.er, or refrigerator *1+ $b for dirty test t$bes *1+ Blea!h *16 blea!h sol$tion to sterili.e test t$bes+ /mall test t$bes *t$bes need to hold a minim$m of 12 m1+ *1 per st$dent+ est t$be ra!( *1 per gro$p+ 0.2,1.2 m1 fliptop mi!ro!entrif$ge t$bes *1 per st$dent+ ransfer pipettes *1 per gro$p+ 0loves *1 per st$dent+ Preparations Before Class: 1. 9$t string into 2.2 ft pie!es.
1

hese tea!her preparation notes, the related st$dent hando$t and other a!tivities for tea!hing biology are available at http)::serendip.brynma%r.ed$:s!i;ed$:%aldron:.

2. /et $p a b$!(et of i!e to (eep al!ohol !old or p$t al!ohol in the free.er:refrigerator $ntil needed. 8. Po$r a small !$p sports drin( for ea!h st$dent. Durin class: 1. Distrib$te !$ps of sports drin( to ea!h st$dent. It is important for ea!h st$dent to s%ish the drin( in his or her mo$th vigoro$sly for at least a min$te in order to obtain eno$gh !hee( !ells. 2. Distrib$te a test t$be ra!( %ith one test t$be per st$dent to ea!h gro$p. Distrib$te one glove to ea!h st$dent. Pass o$t the soap and meat tenderi.er. #lternatively, yo$ !an have a station some%here in the !lassroom %here the st$dents !an a!!ess the soap, et!. 8. #fter at least 10 min$tes *%hen the st$dents have !ompleted the <D"# str$!t$re= portion of the st$dent hando$t+, pass o$t the !old al!ohol and pipettes. 7. #fter 10 min$tes *%hen the st$dents have !ompleted the <D"# repli!ation= portion of the st$dent hando$t+, distrib$te one mi!ro!entrif$ge t$be and pie!e of string to ea!h st$dent. 2. #ssist st$dents> transfer of their D"# to their mi!ro!entrif$ge t$bes $sing the pipettes. It helps to t%irl the D"# aro$nd the end of the pipette to get a large %ad together before s$!(ing the D"# into the pipette. Warn the st$dents to be gentle %hile pipetting so they do not damage the fragile strands of D"#. Ine&perien!ed pipetters have a tenden!y to blo% air into the li-$id and s$!( $p and e&pel the D"# several times in the test t$be before transferring it to the mi!ro!entrif$ge t$be? this tends to brea( the D"# strands. @. P$t on a pair of gloves and !olle!t the test t$be ra!(s from the st$dents. Po$r test t$be !ontents o$t do%n the sin(, rinse the test t$bes, and pla!e them in a t$b of 16 blea!h sol$tion for 10 min$tes to sterili.e them for the ne&t !lass. *Ao$ may %ant to assign this Bob to a st$dent+. 4. Cemove test t$bes from blea!h %ater and invert them in the ra!(s to dry for the ne&t !lass. D. Cet$rn the al!ohol to the i!e b$!(et or free.er:refrigerator. !nstructional Su estions and Additional !nformation

#s ba!(gro$nd for this a!tivity, st$dents sho$ld (no% that D"# is the geneti! material and D"# is !ontained in !hromosomes inside the n$!le$s inside a !ell. EUnderstanding the F$n!tions of Proteins and D"#E *available at http)::serendip.brynma%r.ed$:e&!hange:bioa!tivities:proteins+ provides a s$ggested se-$en!e of a!tivities for introd$!ing st$dents to D"# as the geneti! material. he proteases in the meat tenderi.er not only digest histones *the proteins that D"# %raps aro$nd+, b$t also brea( do%n en.ymes in !ells %hi!h !o$ld digest the D"#. 9old al!ohol helps to pre!ipitate the D"# mole!$les by red$!ing the temperat$re and dehydrating the salty soapy sol$tion of D"# immediately $nder the al!ohol layer. he high salt !on!entration *from the sports drin( and meat tenderi.er+ is also important sin!e D"# mole!$les are negatively !harged and the salt ne$trali.es the rep$lsion among the negatively !harged strands of D"# and allo%s the D"# to !l$mp together. his a!tivity !ombines hands,on e&tra!tion of D"# from h$man !hee( !ells %ith -$estions on D"# str$!t$re and repli!ation that st$dents ans%er d$ring the %ait times for D"# e&tra!tion. For -$estion 8 on page 2 yo$ may %ant to s$bstit$te an alternative hands,on sim$lation version in %hi!h st$dents $se n$!leotide diagram pie!es and tape to !arry o$t D"# repli!ation? templates for the n$!leotide diagram pie!es are provided in the ea!her "otes available at http)::serendip.brynma%r.ed$:e&!hange:bioa!tivities:D"#.

If yo$ prefer to e&tra!t D"# from samples other than h$man !hee( !ells, %e re!ommend the pro!ed$re for e&tra!ting D"# from split peas available at http)::learn.geneti!s.$tah.ed$:!ontent:labs:e&tra!tion:ho%to:. he dire!tions for preparing the mi&t$re of peas, salt and detergent to po$r in test t$bes %ill prod$!e eno$gh mi&t$re for G27 12 m1 test t$bes? then yo$r st$dents !an !arry o$t the steps of adding en.ymes and al!ohol and p$lling o$t the D"#. In o$r e&perien!e yo$ may %ant to $se the blender for slightly longer than 12 se!onds, yo$ sho$ld not be s$rprised %hen yo$ have a lot of leftover peas after the straining step, yo$ !an $se some%hat less al!ohol than s$ggested, and it seems to %or( best if the al!ohol is !old. #lternatively, yo$ !an $se the dire!tions for e&tra!ting D"# from bananas *available at http)::%%%2.le.a!.$(:departments:geneti!s:vge! *sear!h for 0o Bananas++ or %heat germ *available at http)::learn.geneti!s.$tah.ed$:ar!hive:%heatgerm:inde&.html+. #ny of these e&tra!tion pro!ed$res !an be $sed together %ith o$r dis!$ssion:%or(sheet a!tivity abo$t D"# str$!t$re and repli!ation *available at http)::serendip.brynma%r.ed$:e&!hange:bioa!tivities:D"#+. Fre-$ently #s(ed H$estions, including answers to questions about ro$ble,shooting, Understanding the /!ien!e Behind the Protocol, 9omparing the D"# '&tra!ted from Different 9ell ypes, and Ceal,life #ppli!ations of the /!ien!e of D"# '&tra!tion are available at http)::learn.geneti!s.$tah.ed$:!ontent:labs:e&tra!tion:ho%to:fa-.html. Ielpf$l fig$res for e&plaining ho% detergents brea( do%n lipids are available at http)::learn.geneti!s.$tah.ed$:ar!hive:%heatgerm:ba!(gro$nd.html. # D"# '&tra!tion Jirt$al 1ab is available at http)::learn.geneti!s.$tah.ed$:$nits:a!tivities:e&tra!tion: . o help st$dents $nderstand %hy a!!$rate repli!ation of the se-$en!e of n$!leotides in D"# is so important, yo$ may %ant to $se all or part of the follo%ing diagram in yo$r dis!$ssion of -$estion 8 on the top of page 2 of the /t$dent Iando$t. n$!leotide se-$en!e in the D"# of a gene n$!leotide se-$en!e in messenger C"# *mC"#+ (transcription) amino a!id se-$en!e in a protein (translation) str$!t$re and f$n!tion of the protein *e.g. normal hemoglobin vs. si!(le !ell hemoglobin+ personKs !hara!teristi!s or traits *e.g. normal health vs. si!(le !ell anemia+ L$r D"# a!tivity tea!hes st$dents abo$t D"# str$!t$re and repli!ation, b$t in!l$des only minimal dis!$ssion of the f$n!tion of D"#. We re!ommend that yo$ follo% this D"# a!tivity %ith o$r a!tivity EFrom 0ene to Protein ,, rans!ription and ranslationE to tea!h st$dents abo$t the f$n!tion of D"#. his hands,on sim$lation a!tivity *available at http)::serendip.brynma%r.ed$:s!i;ed$:%aldron:Mtrans+ helps st$dents $nderstand ho% the se-$en!e of n$!leotides in a gene spe!ifies the se-$en!e of amino a!ids in a protein %hi!h in t$rn determines the str$!t$re and f$n!tion of the protein and res$lts in !hara!teristi!s s$!h as si!(le !ell anemia. Lne important point that is not mentioned in o$r /t$dent Iando$t is that, d$ring a!t$al D"# repli!ation, sometimes mista(es are made and the %rong n$!leotide is added to the ne% strand of

D"#. D"# polymerase !an <proofread= ea!h ne% do$ble heli& D"# strand for mista(es and ba!(tra!( to fi& any mista(es it finds. o fi& a mista(e, D"# polymerase removes the in!orre!tly paired n$!leotide and repla!es it %ith the !orre!t one. If a mista(e is made and not fo$nd, the mista(e !an be!ome permanent. hen, any da$ghter !ells %ill have this same !hange in the D"# mole!$le. hese !hanges are !alled point m$tations be!a$se they !hange the geneti! !ode at one point, i.e. one n$!leotide. /ome point m$tations res$lt in signifi!ant effe!ts, s$!h as the geneti! disease, si!(le !ell anemia. o ens$re st$dent $nderstanding of the basi! pro!ess of D"# repli!ation, this a!tivity ignores many of the !omple&ities observed in a!t$al D"# repli!ation. #lso, altho$gh the same basi! D"# do$ble heli& str$!t$re is observed in all living organisms *as emphasi.ed in this a!tivity+, the str$!t$re of !hromosomes differs bet%een e$(aryotes and pro(aryotes. For more information on these topi!s, see a !ollege te&tboo( for biology maBors s$!h as 9ampbell, Cee!e, et al., Biology? Freeman, Biologi!al /!ien!e? or Caven et al., Biology.