Department of Health Library Services ePublications

(1)

Department of Health Library Services ePublications - Historical Collection

Please Note: Aboriginal and Torres Strait Islander people should be aware that this publication may contain images, voices or names of deceased persons in photographs, film, audio recordings or printed material.

Purpose

To apply preservation treatments, including digitisation, to a high value and vulnerable Historical collection of items held in the Darwin and Alice Springs libraries so that the items may be accessed without causing further damage to the original items and provide accessibility for stakeholders.

Reference and Research Disclaimer

Please note: this document is part of the Historical Collection and the information contained within may be out of date.

This copy is a reproduction of an original record. Please note that the quality of the original record may be poor and cannot be enhanced with the scanning process.

Northern Territory Department of Health Library Services Historical Collection

(2)

subproject:

DL HIST

595.772

NAN

1989

c)OSo\

D. N. A. probes to distinguish between members of

Anopheles farauti complex

sequencing of the D. N. A. probes and methods to increase the specificity of the probes

Dr Sanjay Nandurkar

M.B.B.S.

I

^COPY ^ONLY

I

DL H !Si ) .-1

2

(3)

00So1/ 1

INTRODUCTION:

Anopheles Farau ti is a major vector of Malaria in Papua new Guinea, the Solomons, and Vanuatu, and the primary vector of Malaria in Australia~

This taxon is kn won to consist of at least 3 sibling species designated as An. farauti No l, No 2~ No 3~ Anopheles farauti No 1 is known to occur in New Guinea, The Solomons, Vanuatu and Australia, No 2 only from Queensland and No 3 in Queensland and Northern Territory of Australia.

These sibling species are identifiable from their polytene chromosome banding patterns or by isoenzyme differences .4

Also An farauti can be distinguished from other two using

to distinguish between

salinity test. DNA An gambia complex. 5

probes have been developed Those probes allowed faster identification and identification of air dried and alcohol stored material.

DNA probes have been constructed for each of the three known species of the Anopheles farauti complex. 6 Such probes can be used for the ecological

and habitat studies required for Malaria vector control. Partial gene libraries were screened for differential hybridisation of total genomic DNA from each species. Probes selected in this manner were effective in distinguishing between the sibling species however there was cross-hybridisation which limited its specificity.

In order to develop more specific probes DNA sequencing techniques were used to sequence the three probes. Work is underway to increase the specificity of the probes by alteration in hybridisation protocols and making oligonuclecotide sequences from each probe.

(4)

1.

MATERIALS & METHODS

PROBE DEVELOPMENT (as done by David Booth)

DNA was extracted from the 3 species of An. farauti using Ish-Horowicz method.7 DNA was partially digested with Sau3A and ligated into the Ban Hi site of the PUC 12 vector and

transformed into the E Coli J.M. 103. Roughly 1000 recombinants of each species were then screened. ( Re .fe-, Dt"'._9Yam 2..)

2. SEQUENCING REACTION a.

b.

c.

6 ugm of the probe DNA was taken and dried through with the help of double stranded sequencing protocol.

(refer Appe~dix)

The DNA was then subjected to the sequencing reaction protocol as explained in sequenase kit (refer Appe~dix)

A 6% polyacrylamide gel was made (refer App~ndix) d. 4ml of soln was loaded in each well, designated G, A, T, &

C respectively. Thus two runs one each of forward and reverse markers were run through.

e. The first runs were done for 1-1. 5 hours and reloading done.

The reloaded gels were also run for the same period.

f. Gel was then dried in a vacuum drier for about 2 hours.

g. It was then loaded into a X-Ray cassette for ovenight exposure (Photostat of a typical exposure ref. dig1)

h. The forward and reverse sequence were read and ambiguities sorted out by repeating the experiment. Clarified sequences were filed into the computer.

i. Using the terminal positions of the forward and reverse

sequences further extensions primers were made for elongation of the complimentary strand. Forward and reverse primers

( oligonecleotides) were synthesised on the DNA synthesizer and then subjected titryl assays using spectrophotometer to analyse the total and stepwise yield. ( Refe,-, Dio.~"t"~M 3.)

2

(5)

The extension of primers is repeated until the forward and reverse sequence thus obtained shows an overlap.

k. The forward sequence upto the overlap and the reverse complimentary sequence of the reverse sequence from the site of overlap constitutes the actual sequence of the probe.

L. The above procedure is repeat_ed for sequencing all the probes.

ooso,

(6)

HYBRIDISATION OF PROBES

DNA was extracted by the Ish-Horowicz method except that after homogenisation samples kept at 65° x 1 hour ( which provided better yield) and then subjected to phenol chloroform extraction steps ( which also

t

ed yield). The supernatent was boiled 98°Cx5min, cooled and then loaded onto nitrocellulose filter using bio-dot apparatus. Equal amounts were added to three filters.

Filters were baked for 2 hours at 80°.

Washing steps from Gale and Crampton⁵were omitted.

PROBE LABELLING

Probe was labelled to 106

cpm/ugm by ramdom priming. However the protocol was changed to include a step where the probe was partially digested with Sau3A X 5mins 37°C linearis the probe.

Filters were incubated for 1 hour at 65°C in Jonshine mix (containing 0. 45 M Na Cl, 0. 45 M sodium citrate, 0. 2% each of bovine serum albumin, Ficold Polyvinyl pyrovlidine, 0.1% SDS, lOmM Hepes pH 7.0, 18 ugm.ml

% kerring sperm DNA and 10 ugm/ml of E Coli tRNa.

'

Overnight hybridisation as per David Booths protocol was not used. Instead mylar sandwich hybridisation technique was employed for 1 hour at

( which was quicker).

A utoradiography at -70°C ( with intesifying screen) was employed.

ooso \

t

(7)

RESULTS

Sequencing Reaction

DNA probes 2, 4 and 10 were sequenced usmg G 6% P.A. gel and the results are as fallows:

Length ( in base pairs)

Probe 2 95 h.p.

163 h.p.

Probe 4

Probe 10 1.2 k.b. (Ref,., 01C\SY01M++s)

Probe 4 in examination shows a 50 b. p. repeat sequence. Probe 2 does not show any obvious repeats. probe 10 has yet to be analysed.

Hybridisation Results

Hybridisation protocols were modified as follows:

1. Change in boil tern. 65° x 1 hour instead of 5 min x 98°C greater lysis of cells more DNA liberated

2. Introduction of phenol/ chloroform step form DNA enhancing the DNA yield.

separation of proteins

Both the procedures increased the amount of DNA available for hybridisation

Hybridisation of the probes 2, 4 and 10 to the total genomic DNA of the An. farauti reveals the following:

1. Probe 4 binds to An farauti No 1 and lesser extent to No 2.

2. Probe 2 binds to An farauti No 2

3. Probe 10 binds to An farauti No 3 and to lesser extent to No 1.

the above results were reproduced at lower stringencies of hybridisation.

Increasing the stringencies in stepwise manner revaled a decrease in the amount of non specific binding and cross hybridisation.

Detailed results are available with the author and not printed m the article.

s-

oo.so \

(8)

DISCUSSION

Sequencing of the probes ' have led us to a better understanding of the probes. Cross hybridisation between An farauti No 1 and No 3 for probe 10 can be explained by the following hypothesies.

1. Probe 10 ( a 1200 b. p. probe) is much longer than probe 4. It may well be that a part of whole of prove 4 sequence may be repeated in probe 10.

2. Probe 4 may not be contained in Probe 10 but that probe 10 may be binding to an altogether different site in the chromosomal DNA of an An farauti No 1.

A clearer picture will be obtained and problems elucidated when probes are scrutinised for simple and complex repeat sequences via a computer aided data search and analysis programme (we do not have such facilities here at Menzies and this would entail that the basic data be sent to Melbourne data bank and then. be linked to our computer).

Our work is now directed towards chopping off the repeat sequences and to shorten our proves i.e. make oligonucleotide probes which have greater specificity.

ACKNOWLEDGEMENTS

I wish to thank the Menzies School of Health Research in general and Dr K Sriprakash and Jon Hartas m particular for helping me to understand molecular biology and assist me in the project.

I also thank Peter Wheelan for his assistance.

ooso, b

(9)

DOUBLE STRANDED SEQUENCING ( A PPc NDI)( A)

Dry

*

6ugm of for each set of reactions set protocol) ( 25 mins in Speed Vac)

Dissolve in 20 ul of O. 2M NaOH, 0. 2 nM EDTA 5'R/T

Neutralise by adding 2ul 2M ammonium acetate pH 4. 5 Total 22 ul

Add x 4 = 88 ul 100% cold ethanol Total llOul

Store in -70°C/20' Spin 10'/12,000 r.p.m.

Remove supernatent

Add 500 ul 70% ethanol RI T Vortex spin 5'/12,000 r.p.m.

Remove supernatent Dry in speed vac

vortex

( *variation from

SEQUENCING REACTION (APPE:NDI)( 8)

1.

2.

3.

4.

Reaction Mix: Anneals template and primer DNA (form above)

Water 7 ul * variation form original protocol

Primer 1 ul

Seq. buffer 2 ul

10 ul

*+ DMSO 10% 1 ul 11 ul

Warm at 65° for 2 minutes

slowly cool to 35° in a plate containing water initially at 65°C Dilute labelling mix 1: 10 ( can be stored long periods at -20°C) Dilute sequenase enzyme 1: 8 ( stored 60 mins - 20°C)

7

0 0 SO\

(10)

5 . Label Reaction Mix Reaction mix (from (1) D.T.T. ^{0. 1 M.}

Labelling Mix ( dil)

s

³⁵ undil 10% DMSO

leave at 20°C for 5'

TERMINATION TREATMENT

10 ul 1 ul

2 ul

1. 6 ul

17. 6 ul

6. Prepare eppendorfs for GA TC for each reaction mix

Place 2. 5 ul of appropriate ddNTP termination mix in each Pre warm at 37°C for 1 minute

7. Tran sf er 3. 5 ul of labelling reaction mix ( 5) in each set of GATC tubes

Mix and centrifuge Incubate at 45°C ^X 5'

8. Add 4 ul of stop solution Mix and store on ice till ready

9. Heat at 75-80°C for 2 minutes and load immediately 4 ul/ run

P.A. GEL POLY ACYLARAMIDE GEL

1. Clean 1 large, 1 small glass plate + 2 side spacers + 1 bottom spacer + comb

2. Siliconise small plate

3. In 600 ml beaker: 42g urea

10 ml 10 X T. A .E.

15ml PA stock 20ml H 2

o

OOSO\

(11)

4. Dissolve

5. Make up ⁾^~^o 100 ml ,

6. Finish plate preparations: Spacers + grease + clamps

7. Filter and degas gel

8 . Make arnrnoni urn pers ul pha te O . 1 g / ml get TEMED from fridge 35 - 40 ul

9. Add ammonium persulphate and TEMED

10. Swirl to mix

11. Pour gel

12. Insert comb and clamp in position and leave it to set with gladwrap

13. After setting remove clamps + comb + bottom spacer

Of

Oo.$0\

(12)

REFERENCES

! . Brian J H ^& Russell, R C (1986) Australian Malaria Vectors Royal Society of Tropical Medicine & Hygiene 77, 278-9

oo t;o\

(0

2. Bryan J H ^& Colluzzi A New Species of the Anophales Complex

Transactions of the Royal Society of Tropical Medicine & Hygiene

..

64. 28

3. Mahon, R J & Miethke, P M (1982) An farauti No 3 a hitherto Unrecognised biological species of mosquito within the taxon

An farauti Laveran. Transactions of the Royal Society of Tropical Medicine ^& Hygiene 76, 8-12

4. Mahon ( 1985) Aust. Journal of Zoology

5. Gale K. R. ^& Crampton J. M. ( 1987) DNA Probes for species

identification of mosquitoes in Ano. gambiae complex medical ^&

veterinary entomology 1, 127-136

6. D.R. Booth, R.J. Mahon ^& K.Sriprakash DNO probes to identify members of An. farauti complex (MSHR)

7. Ish-herowicz D (1982) Rate of Turnover of Structural variants in the r DNA gene family of Drosophila Melanogaster.

Nature 295, 504-568

(13)

S EG VEN C.E"

AS SEEN

"

D iAGr-RAM 1

oF A PAR7 o,=:. A.

OF PRoSE. 10

ON

SoTro,.., OPlo-lA-2-PS READS . .

~ (:.r- C.C. C AA T•"T ~ T >tA-A ••••.

A T

G

., . ~ _ -···- · · - - .'

'X)L.

⁴¹¹⁼

- .. ~-

--~

I

-

4 :

• •

;.....

ooSO\ '1

(14)

Puc /~

VE

CToR

oo.so' ;, 2.

b /Aq-RA M 2.

S"fEP~ IN

DNA pu.-th~ll'f di~~~.,I

,.:;,tt, $t.\u 3C\,

PUC. 1.2. V C:CTOR

01SPLA'II N(;r Vf\-RJOI.JS A.£$1RICT)ON E:NZ'YNE5 SITES.

Puc. ^12. \/Ec:ro(

CUT AT ~ HI SIT'€ .•

(15)

SEcQ Uc°N(.J NC'.{-

E.&...ON~TtoN QF PRIME~ ~LUN~ THE

Te'MPl.ATf'.

~ 6'1, i:>A I M5,C ALON Cr

(OMf'&.I M4NTA~Y ~MP~ta

fe~f.\lNA-L Po~-t1orJ S OF

"' R£t'E"ftSE 'Et.ow~1,0HS o,EJ>~

AS E."'l'ENSIQtJ P~1MEi.S foQ,.

,:V~'Tl-tE R. ELO,.,(';:M.1lON,

O'IF:iJa.c.~f 4'15'1"MNl!O W\Tl-f

" o R w ~ _..,._..c> ~v&iue

f'P.. IMEI\ e">t'T<N $10,-U,

o o S D \ ft'$

YEC'toR

(16)

[ fARAUTI

PROBE

10. FOIJARD

PRIMER]

f:.I:L ^·1^.:.^c.^1_.:¹^• ^'7^... ^ti ^c:^{' ••}^•¹^:^c·^{_ ..}^!

+-VE"C'ToR - - ·~ S i A A i ~

CC i; A Ci CT T ^C:i^f."Jf3 CT Ci C ti (3 !3 'J.' Ci~, ACT CT ti G ,;

(3\G

ti TC C i:3

t';

{i i::i f3 C~ A 13 C "i' 13 J TT CT i.':i TT

TGGGACGTTCCGGGAAATGCCGACGC~CGCGAATGGAACTTAAAATTCC~~CAT

1.-ext pruner

l t

^l

TAAACACCGAACAGGTTGGCT~CAGGCGGTAGATGTT1GTTAATG7GTGGGTAA

~ ACGGTGTCGT1CTT111C. CAfCGCAiCACGGACCAC1GCACACCA

CAATTAlTCCGAACAGCGAGCGTACGTCTTCGGTGGTGCTCTTGATGAAGTA

~

C ⁽¹A {J C ^{(1 1:1}'J '.l_" ·-~·

·r

T [ 'J' '.f 'J.' r\ C

c :

C ;~·! C t! C T C [:

c; ;;

A Ci 'f i\ (-\

ti

^C;T T T T T A ti 1' Ci C CJ Ci A T i3 ( T f3 fJ

-e.xt - - · p..,,.,,· er . 2f~J

, ,.. 'I - . ,.. ·• :-·• ~ ,. 1·· ,. .. r• r·,

r ,·.

^r,,1'

r·· ;, , .

^{'Y' ,-·.}^{r ,}^..··r· f' .. , f' 'J' T f' t' f" T

r· ("•

^f''

C. H 1 L t .. i h (-i L J. H r-'i .•.. ,., 1.:1 .... _i H _;_ _ .:t H P. .1 ,.J i-i H .... :1 l.- ^.J ^.^-·^{.:i _}^..^.:;^. ^{.J . ·}^.:1 1 0 F .... 2 4 .... 8 ....

x: :: "

oOSO\ llf

. ·ATATT1G1GCAAAG111A11TTIGAATGTA1CGAAATGTTGTTGATGAATGGAACGCTTAGG TAAATATITGTATTATTTTACATCATCTTAAGTGAGCTGTCTGTTTTGTTGTAAAGAAATCC

I

?if.':, (1 LT A f-l T f.':'i T (1 CAT(\ Ci{; TC{:; C: C f.:i f3 CC.:

C,

CT Ci

G{ ~

Cc:>ntit1ue.d ~n -rt.Yt.Yse ~~uenc2 .

(17)

u⁰ ~....,, ;

,s

f

FA

1

RA_UT I PROBE l O. REVERSE

PR

!MER]

Gt L J. F> ^a'7 .. C ~::

~v1::,,.~~C-i~A ~C~I~~~

^~-ⁱ^T

ti

C 'j: T 1 ^~1C ~\ C 'ITT {1 TC TT T G G C: TT t-'\ Ci I' T 13 C: C {::, '.f C: 'T TT 1~: l C C1 (:;TC CG Ci C

J e x

t

p r i. Me r-

1

r·

Ci ti T T T T f.3 ,-; C C C A (; G C C T T IJ T T C.:: !.:: 1°3 ;\ C C ,:~ C C T C C T

F t-1 F {;

u

^{T 1 }-'}

r:: u

^{E: }.-;} J.

u

^a r-: ^J.:_;l..,J I:-: }< '.-_ .. _-: _°!"·_·:. t:,~ ' .:.:.· r_i' ·1._ J.. .• • I ~.J. ~-··; ·-,. l_ .. _H_\·.i t:• J. .... _r. ' .. ' 1.· t" .·· .• · J.r_· •• • t[ ·.··. .J,_ I 1j 1· -; -T ·' c:· .._·_1 • C d ^a t.::: '·;. -· .. )

, ... r·:: (':· ')., ,-. 1···. ;-, ,,., r. I- r. n·, r·, ry: r;; r.-, ···. r-:-: I\ (-· A t'- •••• "'\ , ••• :.a.. ··\ .I, ,-.. r:""1 ' -. r.·. ,. .. 1 •• • ... • ., ••• -... r.· -

•.J ' .. ! '··' -· i_. ..J t-i H H H r·i J. J. J. J. l l ... J. t-·1 ^{I ••}- r·( r'• 1 I . -· f.J ^I.. ·1 (-1 • l . - · H ^I-^.J •

1

^f]•• l • l • r· ( ., _ . • •• : ! t-r .. /I i-_. -·· ., I . ,.: .. I H⁰^••• I .... 'l ..· j' . L' . ·.... r··

T

.

c ·

.

,-,

_1 ^1-==_! ^~'•.J ...

r·

· ^1-.::-1 ^{,·. r:·:}L- _l

f"~ f"' f'~ r·· i:; (~ T f"· f"' (: f" ~ f"'

r:· r· ,._

^e^.-4..^r'^'r^f~^f''^•r^f"'^r::^1-·•p ""' r·· ¹r' r· r-[· 1-. r-'"· ·" ,-·. ,. ,-.' ·" ,-. -.. -. ,. ·-. ,,, -. r-. ,. , -•• -.. , ·• ,., , ••.

... .. : ·" · .. .- ... - ., ... .- , .. ·-1 , . .- t"I · .... . J .J h · ... · r 1 • .... . •. ..1 1. .... 1. • .... 1 .. 1 ,.J .1. 1..:i ,_ .. L L· I.J . h l.J rt ri 1_. 1.:i 1...-H 1.., l.:i l ... 1_. l_, H l_j l.J 1-: !.:, ~ -,). 1.... 1.:1

t

^·^QKt, ^·^pv1meY

J-t: ,.,

. .... ·-·.--·, A , . , .. · . . , ,j. ('"\ • I ·: ··- • . • .. I •. • . • . • .

C. L l... I.... 1--i J f.:i (¹f.:i

H

^{l •.}^·C J. A 'J (:;, C f.:i 1.:; 1 C G G l 0i/~

L _ C _ {:,,

(l l f.:: T C G Ci C, C1 (:i Ci T f.:: A T T r_:; C Ci . ]. () J? _....

=~

^{4 -·}

t~

_._...

)'~ :: .

~ O'fc-tl°'p- , i,)yy,IQ"t'c;I SutU(f'Ce_ _ _ .

r-:

_...t'"'. f"·' _{_}_{_J}

r r

_{_1}_{_I}

Tr:

_..._-·^L^{.. :}·1· i": _-·L":

r

_.Ji'" _{-1 -1}

r· -

_t-.

~

f"' _{-1 -}r··:

r: r: r-

_- _-_·_{_1 .J}f"'

r-

_{_,}..., -· -·

'°': .. : r

H ^A

c:

^f''.-·

r·

_1 ^L^··:.

r-

_,

r-

_, ^j~-

r-

.J ^"j''. F .J h A r·• J. -·

r:

1·. · ,· d r.-. l

r:·

.J ^"1^··r t-1 .·

c·

^AT^r;j,. ^~.!", ^r.::_1 ,.,.J. [''PI'; . , J. t·I r ·': I · r' . .!

,-

1' T T T i~

t,

T T

t,

C T C T A C {1 r~ C1 i; (-:, C T T C T t) T C i'.'-i A A A fi T A T C A CC T T T G A A A A CG T T T C 13 T P.1 T T C:1

TGTGCTTAGTTCATTCTGGTGAACCTTA~TCGAATGTTAATAATTTAAATTGACTCACCA

'J.' f3 C:1 ;,

T T T C T T

'J.' A C A

t,

C A ,~ A {, C ,'i G ~1 C A Cr C T C f-~1 C T T A A

r;

^A

T

^fJ^{A T G}

T

^A^(..)~\ A T A A T A C A A

t,

T

A T