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Molecular Genetics 7 777120 Lab 5 Objectives Understand the principles and applications of the polymerase chain reaction Become

me proficient in the technique of the polymerase chain reaction Understand the concept and uses of genetic markers To understand VNTR genetic markers Introduction DNA mutations can arise during DNA replication but are usuasally repaired. o!e"er# sometimes they are not and become pre"alent in a population. As t!o populations di"erge o"er time# they each accumulate different mutations at different sites. $hen sequences "ary bet!een populations# this is referred to as a polymorphism . $e are familiar !ith the "ariation seen in AB% blood groups. This is a protein polymorphism. Variation !ithin the DNA sequence is referred to as DNA polymorphism. There are se"eral different types of DNA polymorphism& R'()* restriction fragment polymorphism RA)D + random amplified polymorphic DNA A'() + amplified fragment length polymorphism VNTR * "ariable tandem repeat# aka ,icrosatellites ,inisatllites -N)s + single nucleotide polymorphism .n this laboratory# !e focus on detection of a VNTR. This !eek and ne/t# you !ill learn ho! to isolate DNA from a cheek s!ab and set up a polymerase chain reaction that detects a VNTR + much like in a crime lab0 .nformation in your te/tbook& )olymerase 1hain Reaction Variable Number Tandem Repeats 6VNTR7 DNA )rofiling pp223*245 p284 pp923*999

Part A: D A Isolation !ro" #uccal cells Materials 4 sterile applicator sticks per person 2m( sterile distilled !ater per person 5.3m( 2, Na% per person 5.3m( 2 , Tris* 1l p 8.3 per person Automatic pipettes ,icrofuge Boiling !aterbath Dra!ing pins -terile 2.3 m( :ppendorf tubes 'loating tube rack .ce Met$od %&or'in( individuall)* 1. )ipette ;55 ( of sterile millipore !ater into a clean 2.3 m( :ppendorf tube# labelled !ith your name or initials.
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9. -crape the inside of your cheek appro/imately 25 times !ith the edge of an applicator stick# and then s!irl the toothpick in the !ater to remo"e the cheek cells. Note: Take care not to scratch the inside of your mouth. You do not need to gouge your mouth, scrape it gently over the surface of your mouth. 4. Repeat the cheek scraping !ith t!o more applicator sticks# s!irling all in the same tube of sterile !ater. The !ater !ill become cloudy and you may see particles floating in the !ater. 4. Add 95 ( of 2 , Na% to the cheek cell suspension to gi"e an appro/imate final concentration of 35 m, Na% . Note: Take care with the 1 M NaO safety glasses at all times. as it is corrosive and !e sure to !e wearing your

3. 1lose the lid of your tube and use one of the dra!ing pins pro"ided to pierce the lid of the tube# this pre"ents the lid opening !hile the tube is in the boiling !ater bath. <. )lace your tube in a floating rack in the boiling !ater batch for 23 min. 7. Remo"e your tube from the boiling !ater bath and allo! it to cool. Add 955 ( of 2 , Tris* 1l p 8.3# to gi"e a final concentration of appro/imately 955 m, Tris* 1l. .n"ert the tube se"eral times to mi/ 6)lace your finger o"er the hole in the lid to ensure none of the liquid is lost7. =. 1entrifuge your tube in a bench microcentrifuge at 24#555 rpm 6ma/imum speed7 for 25 min at room temperature. >. Transfer the supernatant into a fresh 2.3 m( :ppendorf tube. Be careful not to pipette any of the cell debris. (abel your tube !ith your name or initials and store on ice !hile you set up the )1R.

Part #: + ,- P.Materials Automatic pipettes ,icrofuges -terile 5.3 ml )1R tubes %n ice& 5.3m( -terile distilled !ater per person 35?( 25/ )1R buffer per person 93 ( 35m, ,g1l9 per person 93 ( 25m, dNT)s per person 93 ( 35, primer D2-=5' per person& 'or!ard 6D2-=5'7 69=*mer7& 3@*AAAA1TAA11T11AAA1A1TA111A11A*4@ 93 ( 35, primer D2-=5R per person& Back!ard 6D2-=5R7 69>*mer7& 3@*AT1TTATTAAAAATA1A1ATA1111TTA1*4@
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3 ( Taq polymerase per person

Met$od 2. -et up t!o 5.3m( )1R tubes and label one BV2C and the other BV9C# ensure you also label each tube !ith your name or initials. 9. Add the follo!ing ingredients using the post*)1R pipettes& -ea(ent -terile d 9% 25D :/pand igh 'idelity buffer# !ith%UT ,g1l9 ,g1l9 693 m,7 dNT)Cs 625 m,7 D2-=5' )rimer 635m,7 D2-=5R )rimer 635m,7 Taq polymerase ,otal +olu"e ,ube +1 4;.3 ( 3 ( ,ube +2 4>.3 ( 3 (

4 ( 2 ( 5.3 ( 5.3 ( 5.3 ( /5 L

4 ( 2 ( 5.3 ( 5.3 ( 5.3 ( 50 L

3. Add 3( of your DNA preparation to the tube labelled BV2C. ;. 1lose the lids of both tubes. :nsure they are closed properly. 3. ,i/ both of your tubes by flicking the side of the tube <. Ai"e both tubes a brief 6E25 sec7 pulse in the microfuge to bring all the liquid to the bottom of the tube. 8. and your clearly labelled tubes 6ensure your label is on the side and not the lid of the tube7 to the lecturer. $hen e"eryone has finished setting up their reactions# she !ill set the )1R machine going according to the parameters belo!& >;o1 for 25 min >;o1 for 45 s 33o1 for 23 s 6.nitial denaturation7 6Denaturation7

6Annealing7 o 89 1 for 2 min 6:/tension7 Repeat steps b# c and d for 43 cycles 'inal cycle at >;o1 for 45 s# 33o1 for 23 s# 89o1 for 25 min old at 23o1.

8. At the completion of the )1R on the thermal cycler# your sample !ill be stored at *95 o1 until the ne/t lab class.

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u"ber ,ande" -e0eats: -e0etitive D A

The patterns of repetiti"e DNA "ary bet!een indi"iduals and this pro"ides a means to identify if t!o DNA samples are from the same person# related people# or non*related people. -cientists use a small number of sequences of DNA that are kno!n to "ary among indi"iduals# and analyse those to get a certain probability of a match. $ithin a diploid organism such as humans# each indi"idual has t!o copies of a particular gene. The sequences bet!een these t!o copies usually "aries slightly i.e. they are not identical# they are allelic. This is also true for organisms 6i.e. plants7 that are tetraploid# he/aploid etc# e/cept they ha"e multiple copies of each gene. $ithin a population# there may be more than t!o sequence "ariations of a gene that e/ist# e"en though each indi"idual only has t!o of the "ariants. These genes are polymorphic. The sequences that are kno!n to "ary among indi"iduals pro"ide each indi"idual !ith a unique genetic fingerprint. Analysing this is called DNA fingerprinting. -e0etitive D A There are many DNA sequences of unkno!n function that are repeated hundreds or e"en hundreds of thousands of times in the human genome. -ome are typically scattered throughout the genomeF others are repeated in tandem and may occur at only a fe! sites. At a tandem repeat site# the number of repeats "aries !idely in the population# although the repeat number is usually !ell preser"ed during transmission. Therefore each different repeat number can be treated as a separate GalleleG and the site can be treated as a highly polymorphic site !ith multiple alleles. A gi"en person@s repetiti"e DNAs come from the genetic information donated by his or her parentsF he or she could ha"e repetiti"e DNAs inherited from his or her mother or father# or a combination# but ne"er repetiti"e DNAs that neither parent has. -ince repetiti"e DNA patterns are inherited genetically# a gi"en person@s repetiti"e DNA pattern is more or less unique. The more repetiti"e DNA probes used to analyse a person@s repetiti"e DNA pattern# the more distincti"e and indi"idualiHed that pattern# or DNA fingerprint# !ill be. Thus# DNAs from different indi"iduals can be distinguished by analysis of repetiti"e DNA alleles. 1$at is a + ,-2 %ne type of DNA repeat is called a VNTR !hich is an acronym for G"ariable number of tandem repeatsG. Another term for these types of repeats is ImicrosatellitesJ. VNTRs are simple DNA sequences repeated o"er K o"er a number of times in a head*to*tail fashion. They occur at specific chromosomal loci !hich are interspersed throughout the human genome. The number of repeated units "aries bet!een indi"iduals and can "ary bet!een chromosome !ithin an indi"idual 6'igure 27.

'igure 2& -chematic representation of t!o alleles of a VNTR 627.

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VNTR loci are flanked by conser"ed DNA sequences. $ithin these conser"ed sequences# restriction endonuclease clea"age sites occur. $hat these sites are needs to be determined empirically or by sequencing. Digestion of the genomic DNA# separation on agarose gels and -outhern hybridisation using a VNTR specific probe allo!s detection of length "ariation bet!een indi"iduals !ithin a population. The different length "ariants are referred to as alleles. -ince the o"erall length of the VNTR region is small# polymerase chain reaction 6)1R7 can also be used to amplify the entire region by selecting primers that anneal to the conser"ed sequences that flank the VNTR region. The resulting fragments are separated by electrophoresis and do not require a probe for detection# but are "isualised on the gel. Alleles are identified by "ariation in )1R product length.

'igure 9& -chematic diagram sho!ing the position of a VNTR on a chromosome and the relati"e positions of a restriction enHyme site to that VNTR 697.

$here only 4 alleles e/ist for a gi"en VNTR# i.e. a simple case# < genotypes are possible. That is 4 heteroHygous and 4 homoHygous patterns are possible 6'igure 47.

'igure 4& All possible allele combinations that can e/ist !ithin a population !here three alleles e/ist for a gi"en locus 697.

'or n different alleles# there are 6n76nL27M9 possible genotypes. 'or the VNTR !e !ill analyse in this lab 6D2-=57 there are 9> different alleles identified# 955*855bp in length. Thus# the total number of genotypes for this allele is ;43.
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.n a criminal case# ;*< VNTRs are typically analysed. 'or e/ample# !here there are up to 45 alleles resol"ed per locus# up to E;35 genotypes are possible per locus. This means trillions of different DNA profiles are possible for < markers. Nou can no! begin to see ho! a personCs genetic markers pro"ide a unique fingerprint for each indi"idual. o! this great sequence di"ersity is generated at VNTR loci among different indi"iduals is still unkno!n# but could be caused by genetic recombination or errors in DNA replication or repair. 3ses o4 D A 4in(er0rintin( )aternity and ,aternity Because a person inherits his or her VNTRs from his or her parents# VNTR patterns can be used to establish paternity and maternity 6'igure ;7

'igure ;& VNTR analysis pro"ing paternity 697

1riminal .dentification and 'orensics Nou !ill kno! from the many TV programmes dealing !ith criminal in"estigation that a criminal can be identified by his or her DNA. The VNTR pattern of DNA isolated from blood# hair# semen# skin cells etc left at the scene of a crime can be used to assist in the identification of a perpetrator. 'urther# !here the "ictimCs identity is unkno!n# VNTR analysis of their DNA can assist in identifying them.

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'igure 3& VNTR analysis used in a criminal in"estigation

)ersonal .dentification -ince indi"iduals can be identified by their genetic marker patterns# the idea of using DNA fingerprints as a Igenetic bar codeJ to identify indi"iduals has been raised. To store K analyse this kind K amount of information !ould be e/pensi"e and impractical. ,ore established methods such as signatures# .RD numbers# picture .D etc are likely to remain the common !ays to establish personal identification for the foreseeable future. D1560 + ,The D2-=5 locus is located on chromosome 2# the largest human chromosome. :ach of us has t!o copies of the D2-=5 locus# one inherited from each of our parents. Appro/imately =<O of the population is heteroHygous at this particular locus because a different VNTR allele has been inherited from each parent. This VNTR is a 2<bp sequence that can be repeated 2; to ;5 times. This means that the length of the repeats can "ary from 99;bp to <;5bp. There are 9> different alleles of D2-=5. Thus# ;43 different allelic combinations are theoretically possible This locus can be amplified "ia polymerase chain reaction and the products of this reaction run out on an agarose gel. 'ollo!ing agarose gel electrophoresis# the amplified D2-=5 )1R products !ill appear as one band in a homoHygous indi"idual and t!o bands in a heteroHygous indi"idual. 2 , 1 9 ,4 1

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'igure < Agarose gel of )1R products of D2-=5form different indi"iduals. The lanes marked B,C are D2-=5 alleles of kno!n repat number from 2; to ;5# e/cluding 23 since this is rare. The lanes marked B1C are samples of a kno!n genotype run for comparison. (anes B2C# B9C and B4C are three indi"iduals of unkno!n genotype. ,odified from 6;7.

.t can be seen that indi"iduals 2 and 9 ha"e 9 )1R products and are therefore heteroHygotes for the D2-=5 locus. .ndi"idual 4 has one band and is therefore a homoHygote.

Pol")erase .$ain -eaction ,$e Mec$anis" o4 P.The follo!ing material is quoted from ,ark V. Bloom# )h.D. DNA (earning 1enter# 1old -pring arbor (aboratory# 1old -pring arbor# Ne! Nork 2289; The polymerase chain reaction is a test tube system for DNA replication that allo!s a GtargetG DNA sequence to be selecti"ely amplified# or enriched# se"eral million*fold in Pust a fe! hours 6'ig. 97. $ithin a di"iding cell# DNA replication in"ol"es a series of enHyme*mediated reactions# !hose end result is a faithful copy of the entire genome. $ithin a test tube# )1R uses Pust one indispensable enHyme * DNA polymerase * to amplify a specific fraction of the genome. During cellular DNA replication# enHymes first un!ind and denature the DNA double heli/ into single strands. Then# RNA polymerase synthesiHes a short stretch of RNA complementary to one of the DNA strands at the start site of replication. This DNAMRNA heteroduple/ acts as a Gpriming siteG for the attachment of the DNA polymerase# !hich then produces the complementary DNA strand. During )1R# high temperature is used to separate the DNA molecules into single strands# and synthetic sequences of single*stranded DNA 695*45 nucleotides7 ser"e as primers. T!o different primer sequences are used to bracket the target region to be amplified. %ne primer is complementary to one DNA strand at the beginning of the target regionF a second primer is complementary to a sequence on the opposite DNA strand at the end of the target region. To perform a )1R reaction# a small quantity of the target DNA is added to a test tube !ith a buffered solution containing DNA polymerase# oligonucleotide primers# the four deo/ynucleotide building blocks of DNA# and the cofactor ,g1l9. The )1R mi/ture is taken through replication cycles consisting of& 2. one to se"eral minutes at >;*>< degrees 1# during !hich the DNA is denatured into single strandsF 9. one to se"eral minutes at 35*<3 degrees 1# during !hich the primers hybridiHe or GannealG 6by !ay of hydrogen bonds7 to their complementary sequences on either side of the target sequenceF and 4. one to se"eral minutes at 89 degrees 1# during !hich the polymerase binds and e/tends a complementary DNA strand from each primer. As amplification proceeds# the DNA sequence bet!een the primers doubles after each cycle. 'ollo!ing thirty such cycles# a theoretical amplification factor of one billion is attained. T!o important inno"ations !ere responsible for automating )1R. 'irst# a heat*stable DNA polymerase !as isolated from the bacterium Thermus aquaticus# !hich inhabits hot springs.
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This enHyme# called the Taq polymerase# remains acti"e despite repeated heating during many cycles of amplification. -econd# DNA thermal cyclers !ere in"ented that use a computer to control the repetiti"e temperature changes required for )1R. 'ollo!ing amplification# the )1R products are usually loaded into !ells of an agarose gel and electrophoresed. -ince )1R amplifications can generate microgram quantities of product# amplified fragments can be "isualiHed easily follo!ing staining !ith a chemical stain such as ethidium bromide. $hile such amplifications are impressi"e# the important point to remember is that the amplification is selecti"e * only the DNA sequence located bet!een the primers is amplified e/ponentially. The rest of the DNA in the genome is not amplified and remains in"isible in the gel. A00lications o4 P.'ollo!ing the introduction of )1R# the technique spread through the community of molecular biologists like * !ell# a chain reaction. As more scientists became familiar !ith )1R# they introduced modifications of their o!n and put the technique to ne! uses. Almost o"ernight# )1R became a standard research technique and the practical applications soon follo!ed. Not surprisingly# the first applications to lea"e the laboratory dealt !ith detection of genetic mutations. )1R has pro"en a quick# reliable method for detecting all manner of mutations associated !ith genetic disease * from insertions# to deletions# to point mutations. -ome enthusiasts predict that !ithin fi"e years most genetic testing !ill be )1R*based. Duchenne muscular dystrophy is an e/ample of a genetic disease !hose detection has been greatly simplified by the use of )1R. The human dystrophin gene# spread out o"er t!o million base pairs of DNA on the D chromosome# is the largest gene identified to date. Boys afflicted !ith Duchenne muscular dystrophy ha"e deletions in the protein coding regions 6e/ons7 of the dystrophin gene. The gene@s great siHe makes it impractical to e/amine its entire length for mutations# so a technique called Gmultiple/ )1RG is used to sample "arious regions of the gene from one end to the other. The technique in"ol"es simultaneous amplification from nine different sets of primers# all !ithin the same test tube. :ach set of primers is chosen to produce a different*siHed amplification product from a different region of the dystrophin gene. 'ollo!ing amplification# the )1R products are analyHed by gel electrophoresis. Boys ha"ing a normal dystrophin gene !ill display nine different*siHed amplification products# !hile boys !ith deletions in the gene !ill be missing one or more of the )1R products. )1R can also be used to detect the presence of un!anted genetic material# as in the case of a bacterial or "iral infection. 1on"entional tests that in"ol"e the culture of microorganisms or use of antibodies can take !eeks to complete or be tedious to perform. )1R offers a fast and simple alternati"e. 'or e/ample# in the diagnosis of A.D-# )1R can be used to detect the small percentage of cells infected by the human immunodeficiency "irus 6 .V7. DNA isolated from peripheral blood cells is added to a )1R reaction containing primers complementary to DNA sequences specific to .V. 'ollo!ing amplification and gel electrophoresis# the presence of an appropriate*siHed )1R product indicates the presence of .V sequence and therefore# .V infection. The sensiti"ity of )1R is so great that signals may be obtained from degraded DNA samples and sometimes from indi"idual cells. This ability and the inherent stability of DNA ha"e combined to permit DNA to be amplified from some unusual sources# such as an e/tinct mammal called the quaga# an :gyptian mummy# and a three*million*year*old termite trapped in amber. This situation has# almost o"ernight# transformed ignored museum collections of biological specimens into treasure tro"es of genetic information. :"olutionary biologists are using these specimens and )1R to e/plore the genetic relatedness of organisms across species boundaries and no! e"en across time.
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$hen )1R is used !ith degraded DNA samples# it can synthesiHe an amplification product# e"en if the sample@s a"erage fragment siHe is less than the distance bet!een the primer binding sites. During )1R# o"erlapping fragments !ithin the target sequence can function as primers to generate full*length amplifica*tion products. This ability of )1R to utiliHe degraded DNA samples is of great interest to forensic scientists !ho must sometimes !ork !ith human cells in "ery poor condition. The technique has pro"ided conclusi"e identifications in cases !here con"entional DNA typing has failed. .ronically# the greatest concern about the !idespread use of )1R in forensic medicine is the technique@s e/treme sensiti"ity. :"en miniscule amounts of DNA left o"er from pre"ious amplifications can be reamplified# leading to an inconclusi"e result. -e4erences 2. Budo!ie et al. Am. Q. um. Aenet.# ;=&248*2;;# 2>>2 Am Q um Aenet. 2>>2 QanF;=627&248*;;. Related Articles# (inks

Anal)sis o4 t$e + ,- locus D1560 b) t$e P.- 4ollo&ed b) $i($7resolution PAG8. #udo&le #9 .$a'rabort) -9 Giusti AM9 8isenber( A:9 Allen -.. 'orensic -cience Research and Training 1enter# 'ederal Bureau of .n"estigation Academy# Ruantico# VA 99243. Allelic data for the D2-=5 locus !as obtained by using the )1R and subsequent analysis !ith a high*resolution# horiHontal )AA: technique and sil"er staining. 1ompared !ith R'() analysis of VNTR loci by -outhern blotting# the approach described in this paper offers certain ad"antages& 627 discrete allele resolution# 697 minimal measurement error# 647 correct genotyping of single*band VNTR patterns# 6;7 a nonisotopic assay# 637 a permanent record of the electrophoretic separation# and 6<7 reduced assay time. .n a sample of >> unrelated 1aucasians# the D2-=5 locus demonstrated a heteroHygosity of =5.=O !ith 48 phenotypes and 2< alleles. The distribution of genotypes is in agreement !ith e/pected "alues according to the ardy*$einberg equilibrium. 'urthermore# the obser"ed number of alleles and the le"el of heteroHygosity# obtained through the protocol described here# !ere congruent !ith each other in accordance !ith the e/pectation of a mutation*drift equilibrium model for a single# homogeneous# random*mating population. Therefore# the analysis of D2-=5 and similar VNTR loci by amplified fragment length polymorphism 6A,)*'()7 may pro"e useful as models for population genetic issues for VNTR loci analyHed by R'() typing "ia -outhern blotting. -e4erences 2. 9. 4. ;. http&MM!!!.fathom.comMcourseM9285283=Msession2.html . Do!nloaded August# 9553 http& . Do!nloaded August# 9553 http&MM!!!.fathom.comMcourseM9285283=Msession9.html . Do!nloaded August# 9553 http&MM!!!.blc.ariHona.eduMcoursesM2=2ghMrickMDNASprofileMpicture;.html .Do!nloaded August# 955;

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