Experiment 2D is mainly about the Polymerase Chain Reaction or PCR. PCR In the experiment, the collected cheek samples were added with InstaGene matrix prior to lysis. lysis. !his matrix contain contains s Chelex, Chelex, a ne"ati# ne"ati#ely ely char"ed char"ed microscopic microscopic bead which which binds binds or &2 chela chelates tes to cellul cellular ar metal metal ions ions $such $such as %" ' that that are know known n as co(ac co(actor tors s o( en)ym en)ymati atic c reactions. *hen the cells are lysed in the presence o( the chelatin" beads, the co(actors are adsorbed and are not a#ailable to D+ de"radation en)ymes. !he incubation o( the mixture at -/C (or 01 minutes so(tened the plasma membrane, released clumps o( cells (rom each other and inacti#ated en)ymes $e."., D+ses' that tend to de"rade D+. %eanwhile, the incubation at 011/C (or - minutes denatured proteins and ruptured the remainin" cell membranes resultin" to the release o( D+. !he mixture was then centri(u"ed and the supernatant was collected. !he pellet was discarded since this contains the matrix bead that could inhibit the PCR reaction. !he complete master mix was then prepared and was added to the PCR mixture. !his was composed o( the PCR master mix and the PCR primer solution. !he master mix contains the rea"ents needed (or PCR ampli(ication ampli(ication $0' deoxyribonucleotid deoxyribonucleotide e triphosphates, triphosphates, $2' recombinant recombinant 2& !a3 D+ polymerase and $4' %" source. !he !a3 D+ polymerase, an en)yme that could withstand hi"h temperature, was used to synthesi)e new strands o( D+. !he master mix also contains red and yellow loadin" dyes which are responsible (or the master mix5s color and "lycerol which made the samples sink. !he PCR primer solution was used (or it allowed bindin" to one speci(ic site o( the human D+. Primers Primers are short short se"ment se"ments s o( oli"onu oli"onucleo cleotide tides s used used in many biochemica biochemicall and molecular molecular techni3ues such as PCR and D+ se3uencin". !he se3uence o( the primer is complementary to the template strand or tar"et D+ se3uence. !hese primers ser#e as 6starters7 (or the D+ polymerase to extend the tar"et se3uence. In PCR, (orward and re#erse primers are used to (urther increase amplicon yield. !he (orward primer $-5 8 45' attaches attaches and initiates the synthesis in the template strand $45 8 -5', while the re#erse primer $45 8 -5' allows the D+ se3uence extension in the codin" strand $-5 8 45'. Desi"nin" primers (or the PCR is a necessary and critical step (or a success(ul ampli(ication o( the tar"et D+. Certain characteristics o( primers are essential to promote proper annealin" annealin" as well as ampli(ication. ampli(ication. !he primers should be 09 to 2: nucleotide bases in len"th to allow a more speci(ic bindin" se3uence. lso, the se3uence has to contain a hi"h GC content o( around ;1 8 1<. !he relati#ely hi"h GC composition allows that the meltin" temperature is abo#e -1=C. In addition, the GC pairs si"ni(icantly a((ect the meltin" temperature by the e3uation, !m $in =C' > 2$&!' & ;$G&C'. !his e3uation called the *allace rule, estimates the meltin" temperature temperature o( an oli"onucleotide. oli"onucleotide. !he meltin" points o( the primer must be in the ran"e o( around -2 ? 2=C (or double helical stability, and that at hi"her temperatures, the primers tend to (orm secondary annealin" structures and cannot bind with with the the comp comple leme ment ntar ary y stra strand nd.. sid side e (rom (rom thes these, e, the the prim primer ers s adde added d shou should ld not not be complementary to one another since the primer 8 primer interaction would compete with the tar"et se3uence and may result in D+ ampli(ication (ailure wherein the primer was se3uenced rather than the tar"et. @inally, the primer should not be complementary to itsel( to pre#ent and eliminate secondary structures. Aa#in" complementary se3uences in the primer would tend (or the primer to bind with itsel( and pre#ent the interaction with the tar"et se3uence, which leads to D+ ampli(ication (ailure. Care(ul primer se3uences are crucial in the success o( PCR. @orty @orty cycles cycles o( ampli(ica ampli(ication tion were were done. done. Each cycle cycle was as (ollows (ollows B; ᴼC (or 0 minute, minute, 1 ᴼC (or 0 minute minute and 92ᴼC (or 0 minute. minute. !hese !hese temperat temperatures ures were were all crucial crucial B; ᴼC was when when the D+ denatured denatured into sin"le strands, strands, 1 ᴼC was when the primers annealed annealed on either either side o( the tar"et tar"et re"ion re"ion to their complement complementary ary se3uence se3uences s and 92 ᴼC was when when !a3 polymerase polymerase bound and made a complimentary D+ strand to the sin"le stranded D+.
!he ampli(ied PCR samples were then subected to a"arose "el electrophoresis. ut prior to this, PB2 F loadin" dye were added. !he a"arose "el was then stained with @ast last stain. !he re"ion o( the D+ that was used (or this experiment was (rom the PB2 locus o( chromosome 0. !his part has a short sin"le dimorphic lu intron which means that this element may be present in some but not in others. ome people may be homo)y"ous $&H&' $they ha#e the insert in both homolo"ous chromosomes', hmomo)y"ous $?H?' $they do not ha#e the insert in either chromosome' or hetero)y"ous $&H?' $they ha#e the insert in one o( their chromosome'. !he presence or absence o( this insert was detected usin" the polymerase chain reaction (ollowed by a"arose "el electrophoresis. !he primers used (lank both the entire lu insertion $411 base pairs in len"th' and ;0 base pairs o( the PB2 locus to ampli(y a B;0 base pair (ra"ment $i( the lu element is present' or a ;0 base pair (ra"ment $i( the lu element is absent'. "arose "el electrophoresis o( the PCR products is su((icient to distin"uish amon" homo)y"otes $&H&' (or the presence o( the lu repeat $B;0 base pair product only', homo)y"otes $8H8' (or the absence o( the lu repeat $;0 base pair product only', and hetero)y"otes $&H8' ha#in" both the ;0 and the B;0 base pair products.
Figure . Genotype of PV92 locus of chromosome 16. Image source: from http://www.hanoerhigh.us/ /science/rm21"/genetics/p!f/la#s/pcr$alu.p!f
!epartments
Aowe#er, the "el (or the experiment did not turn out correctly hence, the theoretical "el was used (or the discussion.
Figure . %heoretical &ata an! Gel.
Image source: from http://www.hanoerhigh.us/ /science/rm21"/genetics/p!f/la#s/pcr$alu.p!f
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!he hetero)y"ous sample the smaller ;0 base pair band is more intense than the lar"er B;0 bp band. !his di((erence is due to the (act that the smaller (ra"ment is ampli(ied more e((iciently than the lar"er (ra"ment. Copies o( the shorter (ra"ment can be made at a (aster rate than the bi""er (ra"ment, so more copies o( the shorter (ra"ment are created per cycle. In the theoretical "el, students 0, 4 and ; had one #isible band while student 2 had two bands. @or student 2, the appearance o( two bands meant that heHshe is hetero)y"ous $&H?' (or the PB2 lu insertion. !wo students showed homo)y"ous $&H&' (or PB2 lu insertion they were students 0 and 4. %eanwhile, student ; was the only one who was homo)y"ous $?H?' (or the absence o( the PB2 lu insertion. !he #alue o( this lu insert residin" in a noncodin" re"ion o( PB2 is that it does not code (or any protein se3uence and is not related to a particular disease. ecause lu repeats ha#e inserted randomly in humans, the lu insert in the PB2 locus can be #ery use(ul in studies o( allele and "enotype (re3uencies in the human population. !he principles o( the Aardy?*einber" theory can be applied to analy)e "enotypic and allelic (re3uencies o( the lu insert in populations. y determinin" the "enotypic (re3uencies o( the lu "enotype within your student population, the correspondin" allelic (re3uencies can also be calculated. dditionally, the "enotypic (re3uencies o( your class population can be compared to published results o( lar"er population si)es. ecause lu repeats appear in the "eneral population at random, the lu insert in chromosome 0 is #ery use(ul (or the study o( "ene (re3uencies in locali)ed human populations. !heoretically, in some small, "eo"raphically isolated populations, all indi#iduals may be homo)y"ous &H&. In others, the indi#iduals may all be homo)y"ous 8H8. In a 6meltin"?pot7 population, the three "enotypes $&H&, &H8, 8H8' may exist in e3uilibrium. Re(erence Chromosome : PCR *prkshop. Retrie#ed (rom httpHHpchs.pcschools.usHwoad? localHusersHmpur)yckiH PCRJpowerpointJre#iew.pd(. Retrie#ed on 1: Kanuary 2100. Polymerase Chain Reaction Chromosome 0Hlu?tpa2-. Retrie#ed (rom httpHHwww.hano#erhi"h.usH departments HscienceHrm20-H"eneticsHpd(HlabsHpcrJalu.pd(. Retrie#ed on 1: Kanuary 2100. Polymerase Chain Reaction Preparation L mpli(ication. Retrie#ed (rom www.docstoc.comHdocsH...H !he?Polymerase?Chain?Reaction?"el. Retrie#ed on 1: Kanuary 2100. Primer Desi"n. Retrie#ed (rom httpHHbioweb.uwlax.eduHGen*ebH%olecularHse3JanalHprimerJ desi"nHprimerJdesi"n.htm. Retrie#ed on 1B Kanuary 2100.
