The Relative Frequencies of Genotypes for the Alu Insert in the PV92 Region in Population by Polymerase hain Reaction

By Mandy Drake Texas A&M University-Commerce Department of Biology Commerce, TX !"#$

%re&'encies of (enotypes #

Abstract! ) estimated t*e relative and allelic fre&'encies of t*e Al' insert in t*e +,-# region in t*e class at Texas A&M University at Commerce 'sing +C., or polymerase c*ain reaction/ T*e relative fre&'encies of t*e class 0ere /12 for *omo3ygo's dominant, /# for *etero3ygo's, and / 45 for *omo3ygo's recessive/ T*e allelic fre&'encies 0ere /! for dominate alleles, and /"2 for recessive alleles in t*e class/ T*e res'lts are diffic'lt to compare to t*e United 6tate pop'lation as a 0*ole 7eca'se s'c* a small sample 0as 'sed in t*e st'dy/ "ey #or$s8 .elative %re&'encies9 Allelic %re&'encies9 Al' insert9 +,-# region Intro$uction D:A is t*e code t*at controls m'c* of t*e *'man;s appearance, 7e*avior, and cell tendencies/ <ac* D:A strand consists of millions of c*romosomes t*at can tell 's different traits if kno0n 0*at t*ey are 'sed for in t*e 7ody/ C*romosomes are made 'p of alleles, 0*ic* can 7e dominate =>? or recessive =-?/ @*en one is dominate, t*e trait 0ill s*o0 'p even if it is paired 0it* a recessive allele/ )n order to look at one c*romosome specifically yo' *ave to 'se a tec*ni&'e called polymerase c*ain reaction of +C./ +C. allo0s only a single template strand is needed to copy and generate millions of ne0 identical D:A molec'les =Coc*ran et al/, #55$?/ )t allo0s scientists to amplify a specific se&'ence of D:A and st'dy it/ )n o'r st'dy 0e looked at t*e Al' element in t*e +,-# region of c*romosome 14 specifically/ Alt*o'g* t*e origin and f'nction of t*is element is 'nkno0n, 0e do kno0 t*at t*e Al' se&'ence is a7o't 255 7ase pairs long t*at is repeated almost !55,555times t*ro'g*o't t*e *'man genome =Coc*ran et al/, #55$?/ )t is also *'man specific =Comas et al/, #555?/ T*e p'rpose of o'r st'dy 0as to find t*e relative fre&'ency and t*e allelic fre&'ency of t*e class and compare it to t*e fre&'encies of t*e United 6tates/ ) *ypot*esi3e t*at t*e class 0ill 7e comparative to t*e United

%re&'encies of (enotypes 2 6tates 7y !5 percent of o'r class 7eing *etero3ygo's, #! percent 7eing *omo3ygo's dominant, and #! percent *omo3ygo's recessive/ %etho$s an$ %aterials A'r experiment 0as cond'cted on @ednesday, :ovem7er 1", #51# at t*e Texas A&M University at Commerce 7iology la7/ )n order to do +C. correctly and to get a res'lt, it 0as spilt into t*ree parts/ %irst 0e *ad to prepare a D:A template 'sing c*eek cells/ 6econd 0e *ad to do +C. amplification, and t*ird 0e *ad to stain t*e agarose gels t*at *ad o'r +C. samples to get o'r res'lts/ To get o'r D:A sample 0e first scraped t*e sides of o'r c*eeks 0it* a toot* pick, 7eing caref'l not to c't o'rselves 7eca'se 0e did not 0ant 7lood cells mixed in/ After t*oro'g*ly scraping t*e sides of t*e c*eek, t*e toot* pick 0as s0irled aro'nd a micro test t'7e filled 0it* 1ml of 0ater/ @e p't t*e 1ml of 0ater and c*eek cells mix into a centrif'ge at f'll speed for # min'tes/ @e c*ecked to see if 0e *ad a small pellet of c*eek cells at t*e 7ottom of t*e micro test t'7e and t*en caref'lly po'r off t*e extra fl'id in t*e micro test t'7e, 7eing caref'l not to lose t*e pellet/ @e flicked t*e 7ottom of t*e test t'7e to re-s'spend t*e pellet/ T*en 0e transferred #5Bl of t*e re-s'spended c*eek cells into a scre0 cap t'7e containing #55Bl of )nsta(ene matrix 0it* o'r initials on top and flicked t*e 7ottom to mix t*e t'7e contents/ :ext 0e placed t*e t'7es to inc'7ate in !4CC 0ater 7at* for 15 min'tes/ At t*e *alf0ay point of ! min'tes, 0e s*ook t*e t'7e gently, and t*en placed t*em 7ack in t*e !4CC 0ater 7at* for t*e remaining ! min'tes/ @e removed t*e t'7es and s*ook t*em, and t*en placed t*em in a 7oiling 0ater 7at* of 155CC for ! min'tes/ @e removed t*e t'7es from t*e 7oiling 0ater 7at* and s*ook t*em again to re-s'spend/ T*en 0e placed t*e t'7e into a centrif'ge and sp'n it for ! min'tes at 4,555xg to pellet t*e matrix/ @e placed t*e t'7e into t*e refrigerator to cool for 1! min'tes/ :ext 0e *ad to amplify

%re&'encies of (enotypes " t*e region 0it*in t*e c*romosome 14/ @e o7tained o'r t'7e t*at contained t*e genomic D:A template from t*e refrigerator/ @e centrif'ged it for # min'tes at 4,555xg/ @e got a +C. t'7e and la7eled eac* side of t*e t'7e 0it* o'r assigned letter to ass're 0e did not lose o'r D:A sample/ @e transferred #5Bl of t*e s'pernatant from t*e D:A temple, 7eing caref'l not to get any of t*e pellet, into t*e +C. t'7e/ :ext 0e transferred #5Bl of a c*illed +C. master mix into o'r +C. t'7e/ @e placed o'r +C. t'7e into t*e MyCycler t*ermal cycler/ T*e +C. reaction 0as started/ T*e reaction 0ill 'ndergo "5 cycles of amplification, 0*ic* 0ill take 2 *o'rs/ After t*e +C. mac*ine ran for 2 *o'rs, 0e removed o'r samples and s*ook t*em/ @e added 15Bl of +,-#XC loading dye to t*e +C. t'7e and mixed it gently/ @e o7tained a pre made agarose gel, and t*en placed t*e casting tray 0it* t*e solidified gel in it, onto t*e platform in t*e gel 7ox/ @e made s're t*at t*e 0ells 0ere at t*e cat*ode =-? end of t*e 7ox, 0*ere t*e 7lack lead 0as connected/ @e very caref'lly removed t*e com7 from t*e gel 7y p'lling it straig*t 'p slo0ly/ @e po'r approximately # !ml of electrop*oresis 7'ffer into t*e electrop*oresis c*am7er 'ntil it covered t*e 0ells/ @e loaded t*e samples into to t*e $ 0ells, 'sing a clean tip for eac*, of t*e gels in t*e order of 15Bl of MM. =D:A standard? in 0ell 1, 15Bl of Domo3ygo's dominant control in 0ell #, 15Bl of *omo3ygo's recessive control in 0ell 2, 15Bl of *etero3ygo's control in 0ell ", and t*en filling t*e rest of t*e 0ells ! t*ro'g* $ 0it* #5Bl of o'r +C. t'7e mixt're/ @e sec'red t*e lid on t*e gel 7ox and t*en attac*ed t*e electrical leads to t*e po0er s'pply, making s're red 0ent 0it* red and 7lack 0ent 0it* 7lack/ @e t'rned it on and let it r'n at 155, for 25 min'tes/ @*en completed 0e t'rned off t*e po0er s'pply and removed t*e gel from t*e 7ox and placed it into a staining tray/ @e po'red 1#5ml of 155x stain into t*e staining tray to complete s'7merge t*e gel for # to 2 min'tes 7't for no more t*an 2 min'tes/ :ext 0e rinsed t*e gels 0it* 0arm tap 0ater for less t*an 15 seconds/ T*en 0e rinsed

%re&'encies of (enotypes ! t*e gels again 7y placing it in a large container 0it* clean 0ater, gently s*aking t*e platform for ! min'tes to remove t*e stain/ @e repeated t*e rinse t0ice/ T*en 0e placed t*e gels on a 0*ite s*eet of paper to o7serve o'r res'lts/ To find t*e relative fre&'encies for t*e t*ree different genotypes, 0e 'sed t*e form'la of fre&'encyE =n'm7er of people in category?F=total n'm7er in class?/ T*e form'la 'sed to find t*e allelic fre&'ency of t*e dominate allele is pE =fre&'ency of *omo3ygo's dominant people? > G=fre&'ency of *etero3ygo's people?/ T*e form'la to find t*e allelic fre&'ency of t*e recessive allele is &E =fre&'ency of *omo3ygo's recessive people? > G=fre&'ency of *etero3ygo's people?/ Results @it* t*e 'se of t*e +C. tec*ni&'e ) fo'nd t*at in o'r class of 25 people, $ are *omo3ygo's dominate, 1$ are *etero3ygo's, and " are *omo3ygo's recessive in t*e Al' region/ T*e relative fre&'encies are 5/# for *omo3ygo's dominate, 5/45 for *etero3ygo's, and 5/12 for *omo3ygo's recessive =%ig're 1/? T*e relative fre&'encies of t*e Al' genotype in t*e United 6tates sample are 5/#" for *omo3ygo's dominate, 5/!! for *etero3ygo's, 5/#1 for *omo3ygo's recessive =%ig're 1?/ T*e allelic fre&'encies for t*e class are 5/! for dominant alleles and 5/"2 for recessive alleles =%ig're #?/ T*e allelic fre&'encies for t*e United 6tates sample are 5/!1! for dominant alleles and 5/"$! for recessive alleles =%ig're #/? &iscussion @*en looking at t*e agaro's gel 0e *ad to kno0 *o0 to read t*e gel and 'nderstand t*at only 2 distinct o'tcomes are possi7le/ T*e first possi7le res'lt is if 7ot* c*romosomes contain Al' inserts, eac* +C. amplified +C. prod'ct 0ill 7e -"1 7ase pairs long/ T*e second possi7le res'lt is if neit*er c*romosome contains t*e insert, eac* amplified +C. prod'ct 0ill 7e 4"1 7ase

%re&'encies of (enotypes 4 pairs long/ T*e final res'lt t*at co'ld *appen is if t*ere is an Al' insert on only one c*romosome 7't not t*e ot*er t*ere 0ill 7e one +C. prod'ct on t*e 4"1 7ase pairs long and anot*er on -"1 7ase pairs long =Coc*ran et al/, #55$?/ A'r res'lts of t*e relative fre&'encies tell 's t*at t*e most common genotype is *etero3ygo's, so 0*en looking at t*e gel from t*e la7 t*ere 0as a +C. prod'ct on t*e 4"1 and -"1 lines/ 1$ of t*e st'dents *ad only one of t*e c*romosomes containing t*e Al' insert/ A'r middle res'lt 0as *etero3ygo's dominant meaning t*ere 0as a +C. prod'ct on t*e -"1 7ase pairs line/ $ st'dents *ad t*e Al' insert on 7ot* of t*e c*romosomes/ A'r least common genotype 0as t*e *omo3ygo's recessive meaning 7ot* c*romosomes did not contain t*e Al' insert, so t*e +C. prod'ct 0as on t*e 4"1 7ase pairs long =Coc*ran et al/, #55$?/ @*en looking at o'r res'lts 0e can see t*at o'r relative fre&'encies of t*e class compared to t*e United 6tates samples do not matc* 'p exactly alt*o'g* t*ey are close/ T*e *omo3ygo's dominant genotype 7et0een t*e class and t*e United 6tates samples *as a difference of 5/52/ T*e difference for t*e *etero3ygo's genotype is 5/5"/ T*e difference for t*e *omo3ygo's recessive is 5/5$/ ) expected o'r res'lts to 7e similar 7eca'se t*e United 6tates sample is s'c* a large n'm7er of people/ T*e differences in t*e relative fre&'encies can 7e explained 7y 's 'sing s'c* a small data sample, and also possi7le error in t*e experiment/ @*en looking at t*e allelic fre&'encies 0e see t*at dominant allele is t*e most common in t*e class res'lts 7't not 7y m'c* compared to t*e recessive allele/ T*e dominant allele is expected to 7e more common 7eca'se t*e relative fre&'encies are *ig*er 0*en t*ere is a dominant allele in t*em s'c* as *etero3ygo's and *omo3ygo's dominant =Coc*ran et al/, #55$?/ @*en compared to t*e United 6tates allelic fre&'encies, 7ot* *ave t*e dominant fre&'ency *ig*er t*an t*e recessive fre&'ency/ T*e difference 7et0een t*e class sample and t*e United

%re&'encies of (enotypes 6tates sample for t*e dominant allelic fre&'ency is 5/1$!/ T*e recessive allelic fre&'ency difference 7et0een t*e t0o samples is 5/5!!/ T*e difference 7et0een t*e t0o n'm7ers is expected 7eca'se t*e relative fre&'encies are different 0*en compared 7et0een t*e class samples and United 6tates samples/ T*e allelic fre&'ency form'la is dependent of t*e relative fre&'encies/ T*e class sample is representative of t*e United 6tates sample 7eca'se t*e allelic fre&'encies are very similar/ Alt*o'g* 7ot* class sample fre&'encies are similar to t*e United 6tates sample t*ey are not exact/ T*e so'rce of t*e n'm7ers not 7eing exact co'ld 7e 7eca'se t*e si3e of t*e sample 0e 'sed/ @it* a sample of only 25, it does not give a good representation/ Also, t*e experiment 0as an extremely long process 0it* many steps t*at 0ere very easy to mess 'p/ D'man error 0o'ld 7e t*e most prevalent so'rce for t*e difference 7et0een t*e class sample and United 6tates sample/ Based on t*e res'lts of t*e experiment, ) 7elieve t*at t*e most common genotype for t*e Al' insert in t*e +,-# region of t*e c*romosomes is *etero3ygo's/ An t*e +C. res'lt t*ere 0as a line at t*e 4"1 and -"1 lines/ Alt*o'g* o'r class sample 0as small and *ad so'rces of error, ) 7elieve it correctly represented t*e United 6tates general sample 7eca'se t*e res'lts 0ere t*e same/ To get a more acc'rate res'lt ) 7elieve t*at more people s*o'ld 7e 'sed in t*e sample/

%re&'encies of (enotypes $

References Coc*ran, B/, Dees, D/, Hopac*ena, I/, 6lovak, I/ =#55$?/ .etrieved Acto7er 2, #51#/ from Jtam'-commerce/ed'K/

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