12.1-12.2 and 10.3 Flashcards
(19 cards)
12.1 Variation Among Genomes
a stagegring amount of variations exist between gnomes of any two ppl
degree to whcih indivdula human genomes differ form each otehr is becmning apparent
only a small minority of these dna sqeunce vairtaions is repsonible fo rthe phontypic differences that chacerticze indivduals
if certain dna sqeunces have no effect on pheontype tehy are still usefull markers to track gene and chromsomes
extensive dna variation distinuses indivduals within a species
Watson codsiveorved dna double helix, venter was pioner of dna squencing and an anoou genome all showed more than 5.6 mil single nceulotide differences formstadndar human geone
each of the indivdulas diploid genomes contains abt a mil unique dna polymorephisms (alleles) that arent dhared by otehr two ppl while the remianing 2.6 mil polymrpish are shared in the genomes of two or all three idnivduals
whole genome squencing of many otehr genomes verfiies that one avg, the genomes of any two unrelated ppl have abt 3mil sqeunce differences
not only does no wingle wild tu[e human genome seqeunce exits, tehre isnt a thing as a standard wildtype human genome length
deletions, isnertions and duplciations of dna result in genome lengths that differ as much as 1% in healthy ppl
most dna polymorphisms dont infleunce pehnotype
some of the millions of dna polymorphisms between the genomes of ppl must be reposnible for the phneotypc diferences that distinhsuih them as idnivdulas
in reality only small fratcion of dna sqeeunce variatons are nonanymoys dna polymprhsims that can affect pheomtype by altering the nature or amount of one or more proetins or ncRNAs
ex. only abt 5000 of the millions of diferences between these two ppl alter the aa sqeunce of proteins
this makes snese bc: less than 2% of human genoem consist of codons within genes, even when they do occur many muations of codons are silent and if a muation inst sielnt and has delteriosu effects, natrual sleetcion often leads to its disappearence from human population
5000 aa altering mutations, a few thosuedna otehr nonanymous polymrphsims betwene tehse two geonoems likely affect gene expresison like the freqncy of transciption or effifciney of primary trasncirpt splicing to produce mRNa
were still left with consluin that vast majorit of millions of sequnces diferences ebwteen human genomes are anonymous dna polymprmsihs affetcing neitehr the nature nor the amounts of any protein or ncRNA in the body
a difference in watson and venter was in genome that includes CFTR, muattons in which cause cytistc fibrisosis and two other genes
u can see that almost all polymrphsism in theri genomes and indeed in all genoems are located bewteen genes or in introns, consinet with diea that most dna chnages dont akter phenotype
the existence of such vast numebr of ananoymous dna polymprhsims that distingshes different human genomes presnets chalenegs and opportunities
chalnges: how can we sort out millions of polymropshsin in dna to find the few that are relvant to traits like disease?
opportunties lie in teh fact that even if a polymropshsm is annoynous with no effect on pheontype, it still serves as signpost in genom,e a dna marker
researchers can use anonyoms polymrphic loci to help locate the enarby muatins thta are repsonble for inerted diseases
genetic variants occur in severla types
genetsicts usually place polymrphisc dna loci into one of the foru categries based on numebr and kinds of ncuelotide pairs involved
even though the broders bewten clases are fuzy, fateorgies help researchrr descireb what a partcualr genetic variant looks like
there is single nucelotide polumrphsim, deletion insertion polymprsm, simpel sequnce repeat and copy numebr variant
single nucleotide polymrphsim
most common type of genetic variant is single ncuelotide polymproshm or SNPs
SNPs are base positions in the genome where alt letetrs of dna alphabet distinhs some ppl from otehrs
SNPs acount for mjority of total vairtion in human genomes, occuring once every 1000 bases in any pairwise comaprison
mehcnsims that can give rise to SNPs icnldue rare mistakes in DNA rep, or epxosure of genome to mutagenic chemiscals or raidtin in envriment
despite existence of many types of events that can create SNPs, the epr base spont muations rate is still less than on in 30 mill per gen
this is so low that most SNPs are biallelic in human pops with only two of four pssible ncuellide pairs represnetd
the low muatton rate of SNPs allows researchers to trace each dinvdual SNP back to a gnemic change that cocured once in a single ancetral genome
the low muations freq also means that those ppl who didnt inehrtit this chnaged ncuelotide called the deirved allele have a more acicnent ancetral allele that was porb presnet lng before huamn species took form
if a SNP exists, genetcist can take advantage of the close relationship between the human and chimp genomes to dtermine which alelle is ancient and hwich si teh derived alle resultingf roma relavely recent muational event
ex. two single base changes have occured in small genomic region sicne dievrgence of huamn and chimp
one is share dby all human genomes and isnt polymprhic in humans
the seocnd base was form C in chimp to a (derived allee) in a chromsoe of anctro of some ppl but not otehrs
if u and a firned share dirved alel at anomous SNP locus then u got the alle from same ancestor
the fact that every random pair of huamn beings on the planet shared unlned deirved snps allee idnicate comon anctesory for all ppl
anaylys of millions of human genoems has led to ID of mroe than 300 mil SNP loci that aee catgloued in a SNP database
for abt 15 mul of SNPs both alleles are cmmonly found in most human pops
mutaions giving rise to deirved alleles of common SNPs mst have cocured far enough bakc in huam hsirt to ahve been dissemniated to a siganfnt proprtion fo current day ppl
SNP database already incldued a alreg fratcion of all cmomon SNPs in human pops
muation event in very recent past created rare SNP alleles found in one or few of the bilion ppl
for these recent snp alles arnet acounted for in dat abase bc so few human geoems. have eben anaylzed relaive to entire human pop
orgnin of SNPs sugegst that genome sqencing provides pwoerful tools for udnerstading human ancestry
deletion insertion polymorphisms
short insertion or deltions of gentic material rep the seocnd most common form of genetic varton in human genome
thes evarinats are refefredto as deltion-insertion polymorphsims (DIPS) or InDels
while SNP loci occur at abt one per kb in comaprison of two haplodi gonemes, DIPS are considermbly rarer and occur abt once every 10kb of dna
they rnage from lenth fo one base pair to hundgred
freqeuncy decliens iin relation to angel
as reuslt DIPs involing only one or two ncuelodies are the most common
many biochem processes likely contribute to formation of DIPs
tehse icnldue probs in dna rep, recomb and mistaeks that cocur whne cells try to repair dmagae such as brken dna strands
simpel sqeeunce repeats
genomes of humans and higehr eukarotea re loaded with loci deifned by simple sqence repeats, sometimes knwon as microsatelities
SSR loci are suqences of one to few bases rpeated in tandem
diff alles of SSr locus have diff number of repating units
most common repeats are units of 1,2 or 3 base sequences
SSRs with alger repeats are less frqeuent and we employ ehre a relavtly arbiatry cutoff in which the lagest repetaing unit of SSr has 10 bases
ssrs acount for abt 3% of human genome DNA; ssr lcus can be found on avg ocne every 30 kb of human dna
most ssrs occur outside the coding rgeions of genes and have noe fefct on pheonitupe
ssr variation within gens can have rpofund efefct on phoenotye
ex. long trincleotid repeats are moecualr casue of neurlogcial coditons like huntingtons idsease
ssr arise spont from rare randome vents that intally rpodcue a short repeated sqeunce with four to five repat units
once a hsort ssr muates into existcne it can be expande dinto a longer suence by a form of fault dna rep called slipped msiapirg of stuetring
bc of thinsg like mispairng, new allees at ssr loci apppear at rate fo 10^-3 per lcus per gamete
this freq is larger than single nuceltde muationr ate of 10^09 and reuslts in large amount of ssr variation among unrelated dinviduals iwthin a pop
unlike snps- whicha re bialelic and dont chnage after muation event hatgives rsie to them, SSrs are tehrefore higjly plumorphic in numebr fo repeats tehy carry foten qith 10 or more alles distinhsbale at single SSr locus
rare of SSr muation is nonethless low enough that chanegs usually dont occur iwthin a few gens of even a alrge family
u will see that SSrs can thus sevre as relavly stable, highly polymorphic dna markers in linkage studies of many organisms icnldung humans
copy number variants
indivdual human genomes also display dna length polymorphsims involving more than jsut the few nucelotdies charferzing ssrs and dips
researchers were surpsied to fidn that teh genomes of may ppl showing no signs of any gentic disease vary a vairable numebr of copies of alrge blcosk fo gentic material up to 1 mb in length
tehse gentic varinats are reffred to as copy number variaints - CNvs
CNVs are comin in tehri distbution acorss genome and in tehri frucy occur withan hman pops
one of the mosy imprant mechnisms that can prdocue new allees of CNV locus is unqueal corssing over
during meisis 1, tandem arrays of the repating units on homoloous chromsomes cna pair out of rgeister
if recomb takes place bewten mispaired repating units, gametes are prduced that ahve moe eor efwer copies of repetaing unist than orginals
although mehcnsims such as unqeual corssng over make CNV locu highly polympreic, CNVs are still stable when osvred in faimilies oevr few gens: more than 99% of cnv alels in curent human pop are deirved form inertance than new muation
the oflactroy receptor egne family whcihe ncodes proteins that allow animals to smell a dievrse array of odors offers a fascinating exmaple of avriation in gene copy number
typcil mouse geome carries 1400 olfatcory recpetor genes distrbuetd among 50 chromsal sites
but sense of smell isnt needed for surval
olfatcory recpetors then can be lost withoyt consuence and so ppl carry less than 1000 of thsoe genes
but indivudals vary around this mean
some ppl can have hdunreds more or les stahn otehrser reuslting ind fifernces in ppls abilotie sto distingsh odors
12.2 Genotyping a known disease causing mutatin
the ability to genotyep a person for genetic diseases provides lots of helpful info
the ability to dtemrine whetehr a perosn is homozygous or het for a disease causing allele presumes that sicntist known the precise change of ncuelotides repsonble for the disease
for certain disorders like sickle cell diesease teh dientfy of the disease cuaisng muation is clear bc we know how a protein like hemoglobin is aletred in teh disease
but in most cases symptioms dont provide clear info aby teh disease gene
sicistinst find the mutation and once dineiftied indivduals can be genotyped by methods discussed furtehr
PCR amplifies defined regions of genome
determining whetehr a eprson is homygouz or hertozygous for a disease causing alelle or homozygous for normal alallee of teh same gene implies that uc an isolate teh gene from teh eprsons genome and analzye the alleles by looking at the prufied dna
genes are rare targets in complex genomes: the gene for the beta chain of ehmoglobin spans 1400 bases of the 3bil pairs in haploid genome
Mullis inveneted polymerase chain reaction to deal with teh prob of looking for. agene in teh genome
PCR is fast and efficient
it starts with smallDNA like found in a ahri and can make billion copies of a hsort portion of genome in hours
PCR amplifies a atrget region of DNa
two 16- to 30- base long oligonucleotides, the PCR primers define teh ends of the target region
the investiagor syntehsizes tehse primers based on prior knowledge of the genome
one olginocludte is compelmenrty to one strand of dna at one end of the rgeion and the otehr is compelematry to the otehr stand at the oteh end of the region
5’ to 3’ is tehir polarity - arrows point toeahc eahcotehr through target region
for DNA genotyping, the object of PCR is to amplify seuqnces within the target region taht may have diff alleleic forms
the process of amplfictaion is initated by the hybridization of these syntehtic oglincoultdes to one or more dneatured template DNA moelcules (single strand) within the sample of genomic dna to be analyzed
the oglinocluotdes act as a primer that allows dna pol to egenrate new strands of DNA compelenatr to both strands of genomic dna between the primers; rmbr that DNA pol adds ncuelotdies to 3’ end of primer
after time elaspes to alolow copying of teh atrget region, the rxn is heated to melt apart the orginal template strands of dna form teh enwly synstehsized strands
the rxn tube is allowed to cool so that the starting DNA and the copies is syntehsized in the previous step become templates for furtehr rep, using the oglinuclotides remianing in te tube as primers
do this same steps - dentauration to single strands, hybridiztaion of rpimers and polymerization by dna pol - reuslst in epxonenetial inc of numebr of copies of tagret region in eahc step
iterative steps of protocol can be automated in pcr mahcine that heats up and cools smaple to a shcedule
the rxn tubes in mahcine have enough nucleotide tripshates and oligonucleotide primers to support mutlpel roudns of dna rep
tubs contains special dna pol from bacteria that allows it to remain actvie afetr being usbejcted to high temps needed to melt dna strands in pcr protocol
the two oligonoclide priemrs dictate the nature of final pcr products
pcr products are double strands frags of dna that extend form psoition of one primers 5’ end to position of otehr primers 5’ end
the primers need to be compemart to opp strand and have 5’ to 3’ polarities that point twoard eachotehr
pcr is inefficient if primers are far apart so protocol doesnt amplify dna rgeions larger than 25kb
pcr products genotyeps by sqeuncing or sizing
diseases caused by changes incovling one or few ncuelotides in single gene, all the ifno that distingses nromal alleel from muatnt resides in teh genome that can be encomapssed by pcr product
differences ebwteen alelels can be reocg by squncing of the pcr products or incases where muations add or subrtatc nucelotdie pairs form genome, by loking at the size of pcr products
complex polymrhsism like cnv affect many base pairs that extend large rgeion taht cant be amplified so tehy are analzyed bt otehr methods
seqeuencing pcr products
mutation for sickle cell disease changes the dineity of single aa in the chain of teh gemoglobin from gluatmic to valine
the allele is a single nucelotide substitation thatchanges A to T in mRNA like strand of b globin gene; muation is a SNP
by genotypnun alleles of the SNP we can dinefy who has the disease ro are carriers
process begins by PCR amplifying locus from person genomic dna using pair of primers compelemar to sequnce on eitehr side of actyal disease cauisng muattion
once the pcr product is made, the dna sequnce cna be detrmiend by automated sanger method
eitehr one of the two pcr primers can serve as priemr for suenqicng rxns
nucelotide substiytin responible for disease shows up clearly in comapring the sueqnce obtaiend form pcr products in the sickle cell patient, homozygote and het
both alleles are visbe simulayently in the pcr product from a heterozgyotes sequnce of the gene
genomeic dna prep from somatic cell of hetrozygote conatins both alelleic vairants
bc the priemrs hybridize euqall well with the two homologous chromsomes, abt half of the DNa moelcules in final PCr product conatin muatnt squence and otehr half conatin the wildtype seeunce
hetrozygosity for the deiase causing SNP is thus seen as double peak showing both A and T in DNa seunce trace
the etchqnieu fo seqeuncing PCr products amplified from genomic DNa is a straightforward wya to dtermine anyones genotype for any SNP
same method can be used to genotype otehrs kinds of polymorshpms involing small numebrs of ncuelotides like small dleetions or sinsetrions or expansions or contrction of the numebrs of repets in simple seequnce repeats
size variation in pcr products
can also genotype a polymprphsim in pcr product without sequencing it
gel electrospheris can distinhish small variations in size of locus caused by DIPs or SSRs
begin by using pair of rpiemrs compelarty to sequnces on eitehr side of the length polymprhsim to pcr amplfuylocus form idnivduals genomic dna
then u subject pcr products to gel electrophreiss which seps them by size
after staining with ethidium bromide, each allele appears as a specific band of dna
the ability to genotyep size variants in this way is impritant for the egnic diseases caused by trinucleotide repeat SSRs within genes
Huntingtons is a trincuelotdie repat in huamns
autosmal dom
symptoms show up ealrier in age if more repeats
36 or more repats of CAG
expresitvy is variable by with 42 or more its compelty penetrant
some ppl with HD histroy would like to know their genotype before deicing to have children
by amplfying the CAG conating part of the HD gene using priemrs that flank the SSR and emaising lenths of PCRporudcts, genetcist can dtermien how many cAG eepats were found in eahc alelle
fetal and embryonic cells can be genotyped
couple expecting baby knows by egnotyping their genome they are carries of alele fro delterious recieve gene or that one has a dom allele for late onset condition
some parents would prefer to terminate pregancy if they knew fetus had teh disease causing genotupe
prenatal egnetic diagnosis involves genotyping fetal dna
physicans can isolate fetal cells by amniocentsis or obtain fetal dna in motehrs blood using non invasive prenatal testing
genetcist pcr ampfy the disease locus form fetal dna and then analyze pcr products by seuqencng or sizing
success of in vitro fert and pcr have opened mroe options
its posible for couples toe stbalish genotype of embyro before palce din motehr
preimplantion embyro diagnsosi begisn when female is inejcied with follcile stimulating hormone to stimulate matruation of 10 eggs
obsterican removes eggs and fertlzies them in vitro with partenrs sperm
fetrlized eggs are incudbated allowing mitotic diviso to get 6-10 cells of emrbyo
then a single clel is removed from each embyo - they arentd etrmiend to become cells yet so removing doenst affect embryo or proepr devlopment
tehcnician tehn rpepares genomic dna from cell and pcr amplfies the rgeion of muation
they then nalzy epcr by seuncing or sizing
parnets can slect healthy emrbyos with genotypes that wouldnt reuslt in disease (homzygote for normal or het)
two or three embyros are then palced in mom to imrpvoe chances that one sucesfuuly implants
that embyros can be genotypes within a few days after fert by looking at dna form single cell is astonishing but preimplant embryo diagnsois has been sucesful in tens of thosuands
complex and expresive preocedure but invaluable to those who coulve pased down serious diseases
10.3 Sequencing DNA
the petri dish with thousedands of sep colonies constitating prt of human genomic library
each clone contains a diff recomb dna moelcule eahc with a plasmid vector atatched to a diff frgament of human genomic dna
the arrangement of them on a plate had no order in genome
if teh library is made with oevralpping genomic dna frgas we can sqeunce the human genome frag in each clone and use this info toa ssemble the base pair saaeunce of each human chrosmoem
dna sequncing tech was based on sangers method
his method can be autamocted providing eficney to seeuqnce the 3 bill nucelotides in human genome
sanger seuqenicng depends on DNA pol
sanger based his tehcnique on knowledge of way dna is repplciated in cells
enyzme dna pol catlyzed dna rep
teh enzyme needS: a tempalte - single strand of dna to copy, deoxyribuncuelotide triphosphates which are building blcoks of newly synethzied dna and a primer that is a hsort single stranded dna moelcules (an olihgonucleotide) thats compelenart to part of template and that provides teh free 3’ end to whcih dna pol can attach new nucleotides
to sqeunce DNA using dnagers method u need a template, part of whosse sequence in known but remainder is unknown bc thats what ur tryng to detrmine
one strand of teh dna of a recomb plasmid could serve as the template - u know the dna sequnce of teh vector but not of the genomic dna insert
next u need a short oligonucleotide primer comprlenart to the known sequence of the vector just adjacent to teh unkwon human dna insert
primers are made to order in DNA synethzier mahcines that can manufactore large qunatities of any dna oligonuceoide up to 200 bases in length
user simply types the desired sequnce of ncuelotides into compuetr controlling syntzher and mahcine then strings those nuceotdies toegtehr in proepr order using chem rxns
u can deisgn the rpiemr bc u already know the sqeunce of teh vector which was detrmined by alt chemical technqiues that dont require prior knowledge
sanger quencing allows the template and primer to inetract through process of hybridization: natrual tendecny of compemanrty single stranded moelcuales of DNa or RNA to base pair
to make teh tempalte u could amplify and prufiy double stranded recomb dna form one clone and then melt the dna into single strands by raising temp to disrupt h bodns that would keep teh strand toegtehr
even though both strans of dna frag are present ind na sample, only one is sued for template in seuncing
u mix the large lamounts of single stranded primer then
as temp of mixture is lowered, h bodns form between compelar nucelotdies of primer and template of reocmb dna
the prmer made has to be long enough to hyrbidize stably - anneal - only with one comeplary seunce in template - priemrs are 17-25 bases
inetratcion of primer and tempalte creates substrate for action of dna pol
sanger sqeuqencing generates nested set of dna fragments
to reveal the order of base pairs in isolated dna moelcule, snager sequencing uses dna pol to create series of single stranded frags that differ in length by single nuceloide
within the graduated ste of frags knwon as an nested array, each fragment ahs the same 5’ end but frags differ in length by number of cnelodes added to 3’ end
it can also disting what base is presnet at 3’ end
so the lnegth and idneifty of 3’ base distinshhes frgaments
sqeuncing procedure to create nested array begins by adding dna pol to the annealed template and primer along witha carefully calibrted mixtrue of 8 ncuelotide triphspahtes
4 are normal dNTPS and otehr 4 are unsal and added at low concnetrations - tehse are dideoxybribnucltde triphapstes - ddNTPs
the ddNTPS lack teh 3’ hydrbxyl group crucial for formtaion of the phosphidester bodns that extend the chain during dna pol
each ddNTP is labsed with diff folour clurocent tag
seqeuncing rxn tube conains billions of identfcal hydbrid dNA moelcuels in whhc the oligonucelotdie primer has hyrbdized with teh template dna strand at teh same location
on eahc moecule, the primer supplies a free 3’ end for the dna chain extendion by dna pol
pol adds ncuelotides to strand
until, by chance, a ddNTP is incropated
absence of the 3’ hydorxyl group in ddNTP prevents the DNA pol from forming a phosphdietser bond with any otehr ncuelotide,e ending the pol for that new strand of DNA
when rxn is done, new strands are relased from template by denaturing dna at high temp
result is a nested set of dna stranded dna frags that have same 5’ primer end and diff 3’ ends
length and colour dtermined by final ncuelotide added
dna frag fluorescence revelas the nucleotide seequnce
researchers analyze the mxitrue of dna frags created by the seqeuncing rxns. trhough polyacrmlide gel electropheresi allowing sep of dna moelcules diffeirng in single nucekotide lnegth
gelk is exmained by dna seqeuncing machine that has a laser to ativtae the ddNTP flueorcsnet tags and a sensitve detctro that can distings the colours
as each dna frag passes the alser it glows in one colour dictated by the dye attached to that end
each signal reps a chain thats one nucelotid eloneg rthan the prev
detector trasnmits info abt signals to compuetr which shows as diff colour peaks
compuetr in dna sqeuncing machines have base claling software the interprets peaks as bases and generates digital file called a read of the sequences of bases compsing the dna
this is compelnart to that of the template strand udner analysis
dna sqeuncing machines can dtermien abt 700 to 1000 bases form any single sample
tehse machines can also run hudnreds of smapels in paralelel on sep gel lanes, each recorded with sep fluroescence detctor
thus a single machine running for few horus can dtermine hudnreds of thosudands of basees of dna seuqnce info
