Analyzing haplogroups through individual mtDNA extraction and sequencing By Dan Lordan

Introduction: Human cells contain both nuclear genomes and mitochondrial genomes. These genomes give clues to our ancestral lineage by sequencing our DNA !e can identi"y single nucleotide polymorphism #$N%& di""erences !hich can distinguish our ancestries "rom others. mtDNA is passed do!n "rom mother to child' so by isolating and sequencing our mitochondrial genome !e can "ollo! our lineage on our mothers side all the !ay bac( to )*itochondrial +ve', our common maternal lineage. -n this lab the section o" DNA used !as Hypervariable .egion /' a region in the mtDNA that is prone to variation. By identi"ying the di""erences bet!een the sequenced DNA and the 0ambridge .e"erence $equence' haplogroups can be identi"ied. These haplogroups have been plotted on an mtDNA phylogenetic tree. The 12 main branches mtDNA' o" !hich there are 12 ma3or di""erent branches' trac( the divergence "rom the mt4*0.A #mitochondrial most recent common ancestor&. There"ore' by "iguring out to !hich haplogroup an individual belongs' !e can identi"y the ancestral migration "rom A"rica.

Materials and Methods: All materials and methods "rom procedure given by )A *olecular -nvestigation o" Human 5enetic 6ariation, as laid out by the B-7 189* course. No deviations "rom the original plan !ere made. DNA !as sequenced at the 0ore Nucleic Acid :aculties at

The %ennsylvania $tate ;niversity. *+5A !as the program used to study and compare to r0.$ data #given "rom B-7 189* course&. DNA !as "irst extracted using a chee( s!ab. -t !as then isolated and H6- %0. ampli"ied using the primer sequences L/<==24 0T00A00ATTA50A000AAA50 and H/2>9/4 T5ATTT0A055A55AT55T5 as !ell as Taq polymerase. The %0. products !ere then run on an aragose gel using electrophoresis and photographed. The DNA !as then puri"ied and placed on a sequencing plate to be sent to the 0ore Nucleic Acid :aculties. :or "urther speci"ics please see outlined procedure.

Results and Discussion

0ontrol John John Dan Dan Ladder

Gel electrophoreses showing DNA from John Lin and Daniel Lordan against a DNA ladder and control

Alignment Reference Position Sequence Position

Mutation rCRS to your sequence

Is this a known polymorphism?

Haplogroup Designation s! for this site tentati"e in#icate# haplotype in

1<9 1A< 88B

/211> /21<= /28//

T to 0 0 to T T to 0

?es ?es ?es

$ol#! N/R Subgroup: Uk #"ormerly (no!n as @& Not available * $ubgroup C' N/R Subgroup: Uk

Table 1: Haplogroup designation from sequenced DNA As per the given procedure' the 8D end o" the L sequence #0T00A00ATTA50A000AAA50& corresponds to position /<==2 in the re"erence sequence. $ince this sequence begins at position 11 in the sequenced alignment' each nucleotide position in the sequenced alignment can be expressed as /<==2411 E#position o" nucleotide& or /<=B> E #position o" nucleotide&. This is the number listed under ).e"erence $equence %osition, in the Table /. The DNA that !as sent to the lab came bac( inconclusive' due to possible errors in %0. or ampli"ication. -t is unclear !hat problem occurred' but it must have occurred be"ore being trans"erred to the lab because the gel had no clear bands #!hich !ould imply that no DNA !as pulled through in electrophoreses&. There"ore the sequenced DNA !as that o" my TADs' Dre! :ister. Fhen using *ito!eb it !as discovered that the H6$/ Haplotype *ar(ers corresponded to the NG. haplogroup' and' in particular' the ;( subgroup. The ;( subgroup is relatively ne! as compared to other groups' and easily distinguishable #.ichards et. al' 1999&. There are only three ma3or places that the H6/ sequence !ould be di""erent "rom the r0.$ data and t!o o" them !ere "ound. Fhile base pair /21<= !as unidenti"iable' it is not concerning that the third nucleotide position !as not "ound. .ichards et. al "ound that /2=98' the other spot that distinguished subgroup ;( "rom other NG. haplogroup subsets' !as a ne!er mutation. There"ore the subgroup ;( could

contain /2=984/211>4/28// as !ell as !hat !as sequenced here' /211>4/28//. The /21<= is slightly con"using' but since it !as not a speci"ic site that identi"ied any other haplogroup or subgroup' it is probably only a mutation. ;sing the identi"iable regions' /211>4/28//' it is sa"e to assume that this sequence came "rom an individual o" NG. subgroup ;(. ;sing the Forld *igration *ap' the ;( subgroup can be traced coming "rom Northern A"rica' into the *iddle +ast' and the bac( to!ards Festern +urope. -t is unclear i" this is consistent !ith the "amily history o" the individual sequenced as that in"ormation is not available. Fhen a BLA$T search !as conducted the H6/ sequence came up !ith =AH accuracy against its closest match' )Homo sapiens mitochondrion' complete genome., There !ere 2 mismatched pairs #8=8G8=2 correct& including 8 near the end in !hich the data !as inconclusive. The other 8 matched up !ith the nucleotides that !ere "ound previously' against the r0.$ data. The sequence came "rom a paper by Andre!s et. al' !hich !as entitled ).eanalysis and revision o" the 0ambridge re"erence sequence "or human mitochondrial DNA., This paper corrected // nucleotide sequences "rom the original r0.$ and con"irmed another B.

References:

/. ;nderstanding Haplogroups' )Deep Ancestry,. Genebase Tutorials. .etrieved *arch /=' 19/>' "rom http GG!!!.genebase.comGlearningGarticleG8A

1. Hass' 0.A.' and @. Nelson. 19/>. A molecular investigation o" human genetic variation. -n A Laboratory *anual "or Biology 119F %opulations and 0ommunities. #Burpee' D. and 0. Hass' eds.& Department o" Biology' The %ennsylvania $tate ;niversity' ;niversity %ar(' %A.

8. .ichards' *artinI *acauly' 6incentI et. al. 1999. Tracing +uropean "ounder lineages in the Near +astern mtDNA pool. -n AJ5H 6olume 2B -ssue <.

Andre!s' ..*.' @ubac(a' -.' et al. /===. .eanalysis and revision o" the 0ambridge re"erence sequence "or human mitochondrial DNA. -n Nat. 5enet. 18 #1&' />B /===.