Tony Corcoran
Analysis of amplified DNA sequences and virual posiional clonin!.
Mitochondrial DNA is for the most part conserved between individuals, but contains
hypervariable regions. Using restriction enzymes which contain recognition sites which lie
within the hypervariable region, we can assess the frequency of variability by observing the
restriction fragments which result from digestion. he restriction enzyme used in the
e!periment is Alw""#, and it is e!pected that the number of restriction fragments produced
will vary between individuals. A successful cut will result in the production of $ small
fragments, and a longer %original uncut& fragment.
'n the second part of the practical, human genome databases are e!plored in order to
investigate the candidate gene for an inherited disease.
$e%ods# As per manual
A' Analysis of amplified DNA sequences
' ran my DNA on two separate gels, as ' only added #()l DNA in gel ".
*el +
'n the above sample, my DNA is in lane ,. My DNA is uncut by the Alw""# restriction
enzyme indicating that the D-loop does not contain the Alw""# restriction site. An e!ample
of a successfully cut DNA sample can be seen in lane $. he resultant fragments are
appro!imately ##((bp and +((bp long.
*el "
'n the above sample, my DNA is in lane .. My DNA is uncut by the Alw""# restriction
enzyme indicating that the D-loop does not contain the Alw""# restriction site. 'f there were
more DNA present it would be easier to see the presence of smaller fragments if present.
(' )irual posiional clonin! e*ercise.
he /nsembl database began in #000, with the aim of annotating the human genome. 't
provides a collection of genomic and biological data, so that a full record of individual genes
and their charachteristics is available.
/nsembl allows us to search the human genome for the given mar1ers %D2340"0 and
D230,..& and to identify the genes in this candidate region. he list of candidate genes can
then be searched on ensembl or other online genomisc databases for more information on
mutations, orthlogues, paralogues and associated diseases.

+rovide a lis of all ,no-n !enes -i%in %e re!ion delineaed .y flan,in! mar,ers.
526#, 78#D$., 9A2, *:;D., <7M=, 2U>=0?#, *AA#, @;<8N, 9D<#=, /7@,
Give a one/line descripion of %e funcion of %e correspondin! !ene producs.
@rotein encodedA @utative ribosomal <NA methyltransferase
@rotein functionA 7inds 2-adenosylmethionine, the methyl donor for
methyltransferase reactions in DNA and <NA synthesis.
@rotein encodedA
@rotein functionA <ab-*@ase activatiing protein.
@rotein encodedA 9is1ott-Aldrich syndrome proteins. 8ytoplasmic proteins
e!pressed e!clusively in haemopoietic cells.
@rotein functionA 2ignal transduction for cell membrane to cytos1eleton. 7inds
Arp$B= comple! to allow polymerization of actin.
@rotein encodedA *lyo!alase domain-containing protein .
@rotein functionA
@rotein encodedA @utative <NA-binding protein =
@rotein functionA /nhancement of protein synthesis in hypothermia, and phosphoryaltion
of translation initiation factors
@rotein encodedA ?istone-lysine N-methyltransferase
@rotein functionA
@rotein encodedA /rythroid transcription factor
@rotein functionA ranscriptional activator which may trigger development of
@rotein encodedA @robable protein-cysteine N-palmitoyltransferase porcupine
@rotein functionA Modulates the processing of 9nt proteins. @robable protein-cysteine
N-palmitoyltransferase adds palmitic acid to cysteine residues of the
9nt family members.
@rotein encodedA 9D repeat-containing protein #=
@rotein functionA 'nvolved in formation of protein comple!es which affect processes
such as cell cycle progression, signal transduction, apoptosis, and gene
@rotein encodedA =-beta-hydro!ysteroid-Delta%,&,Delta%+&-isomerase
@rotein functionA 8atalyzes the conversion of Delta, sterols to corresponding Delta+
1u!!es one <or several' candidae !enes you %in, could .e .e%ind %e disease
5A1 and GATA3.
C%aracerise %is !enes<s' fur%er.
:GNC 1ym.ol# 5A1
Gene /N2*((((((#.$,.
6ocaion 8hromosome 3A ",,.=",0,.-",,."0,,#,
Gene ype Cnown protein coding
1rand 5orward
Analysis /nsemblB?avana merge gene
@araloguesA 9A2:

;rthologuesA Dog %0#D orthology&, mouse%,+D orthology&, zebrafish
%4(Dorthology&, fruit fly %=+D orthology& and ba1erEs yeast.
MutationsA 3-lin1ed recessive immundeficiency characterised by eczema,
thrombocytopenia, recurrent infection and bloody diarrhoea.
1N+ "D )alid C%r = pos Nucleoide sequence Amino acids encoded
-- ",."#$""%F&
**88A88*8A B
**88888*8A >@@3
8,5 ",."#0(#%F&
A*888*8A8A B
A*8888*8A8A 2@A3
8,?, ",.""4.(%F&
8*A*8****8 B
8*A*8A***8 <A*3
-- ",.",00,%F&
8*88* B
1C1= > 151=
8 ",.""4((%F&
***8*AA* B
***88AA* GA?= / 1op
-- ",.""404%F&
**8**8A*A* B
-- ",.".""=%F&
***8A8A**8 B
G:$= > G@$=
-- ",."+##$%F&
A*8***** B
A****** G8*3
8 ",."00(+%F&
A*A8A8 B
A*88A8 0&6= > 016=
-- ",."0=,(%F&
A88AA8 B
T6T= > T6N=
Analysis of the DNA e!tracted in the previous practical showed that the DNA remained
undigested by the restriction enzymes as only one band at ##((bp was formed in both cases.
?ad the restriction enzyme cleaved the DNA at any site, there would be additional bands
present. 't is clear from observing other results from the class that the number of restriction
sites varies between individuals as some gels contained $ or = bands.
' have identified 9A2 as the candidate gene causing the disease described. ' have found
sufficient evidence that mutations in the 9A2 gene are associated with 3 lin1ed recessive
immunodeficiency, the characteristics of which are eczema and thrombocytopenia H those
same symptoms described in the brief. ;ut of ten variations in the nucleotide sequence of the
gene, ' have identified " which result in altered peptide sequence, and one which codes a stop
A full blood count can be conducted to confirm thrombocytopenia. A normal person will have
#.(((( H "((((( plateletsB )l blood. A count of lower than #.(((( plateltes B )l blood
indicates thrombocytopenia.
'n order to confirm that variations in the 9A2 gene are responsible for the disease observed '
propose several further stepsI
#. Assemble a group of unrelated patients and sequence the coding e!ons and splice
sites. 2creen these sequences, and loo1 for nonsense mutations, splice-site mutations
and frameshifts.
$. 'f mutations of 9A2 orthologue can be observed ba1erJs yeast or drosophila, <NA
interference techniques can help identify the relevance of this gene. Cnoc1down of
the 9A2 orthologue gene in wildtype models should result in the mutant phenotype.
=. 8hec1 for relevant mutant dogs, or create a transgenic model in a dog. he phenotype
of an affected dog should be similar to the human diseased phenotype.
T. 1rac%anA A. &ead / :uman molecular !eneics 4
T. 1rac%anA A. &ead / :uman molecular !eneics 2nd ed.
www. ensem.l .orgB
Gou missed a few questions from the manual.
Gou needed to wor1 out what percentage of students contained the restriction site %out of
those that showed any bands&.
Gou also needed to show where in the sequence provided %from practical one& would you find
the Alw""# restriction site H thatJs Kust copy, paste and highlight the **8A8.
Gou needed to describe briefly the approach you too1 to identify your gene of interest.
Gou were as1ed to say how many transcripts there are for each gene.
LLGou were as1ed to list = disorders associated with 9A2. hese are 9is1ott-Aldrich
syndrome, thrombocytopenia type # and !-lin1ed severe congenital neutropenia. 'Jve given
you " out of . mar1s for this.
5ollowing lin1s until you get to
will show you all the tests available.

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