400
Specialia
EXPERIENTIA 32/3
A DNA Extraction Procedure Practicable Under Field Work Conditions
j.
SCHMIDTKE 1
Insr /i~r Humc~ngenetik und A nthropologie, Albertstrasse 77, D-78 Freiburg i. Br. (German Federal Republic, B RD), 3 October 1975. Summary. A D N A e x t r a c t i o n p r o c e d u r e is d e s c r i b e d w h i c h is d e s i g n e d t o b e used in field work. A c r u d e i n i t i a l cell p r e c i p i t a t e , w h i c h is easily o b t a i n e d , m a y e n d u r e s t o r a g e of m o n t h s . A p r o c e d u r e is d e s c r i b e d w h i c h allows one t o p r e p a r e D N A f r o m b l o o d of v e r t e b r a t e s w i t h n u c l e a t e d e r y t h r o cytes, a n d w h i c h , i n t h e i n i t i a l stages, r e q u i r e s o n l y t h e simplest technical equipment. The method has been d e v e l o p e d for field w o r k purposes, a n d h a s t h u s f a r b e e n a p p l i e d t o a large n u m b e r b o t h of f r e s h w a t e r a n d seaw a t e r t e l e o s t e a n fish, a n d to a v a r i e t y of a m p h i b i a n s . T h e p r o c e d u r e is e x p l a i n e d h e r e w i t h b l o o d of t h e r a i n b o w t r o u t (Salmo irideus) as a n e x a m p l e . As a c o n t r o l , a c o n v e n t i o n a l D N A e x t r a c t i o n f r o m nuclei isolated f r o m e r y t h r o c y t e s d e r i v e d f r o m t h e s a m e a n i m a l was performed. Methods. 10 d r o p s of b l o o d f r o m t h e c a u d a l vessels were collected in 10 m l 0.1% h e p a r i n - 0.55% NaC1 a q u e o u s s o l u t i o n ( a b o u t 105 cells/ram3). T h i s s u s p e n s i o n was h a l v e d , a n d one h a l f p r o c e s s e d i n t o nuclei. F o r t h i s p u r p o s e t h e e r y t h r o c y t e s were i s o l a t e d b y c e n t r i f u g a t i o n (10 rain, 200 • s u s p e n d e d in 20 m l of R i n g e r ' s A m p h i b -
E .800
!
%
$
t
.600
/
.400
~L
t~
-j
.200
t a n s o l u t i o n c o n t a i n i n g 0.5% glucose, a n d t h e n lysed with Triton-X-100 (Serva; final concentration 0.15%); a n o t h e r 20 m l of R i n g e r ' s a m p h i b i a n - g l u c o s e s o l u t i o n was i m m e d i a t e l y a d d e d to t h e h e m o l y s a t e , f r o m w h i c h nuclei were s p u n d o w n a t 3000 • i n a S o r v a l l centrifuge. T h e second h a l f of t h e o r i g i n a l b l o o d s u s p e n s i o n was p o u r e d i n t o a 100 m l E r l e n m e y e r flask a n d t r e a t e d as follows: A 4~o T r i t o n - X s o l u t i o n was a d d e d u n d e r m o d e r a t e s h a k i n g u n t i l a g e l a t i n e o u s s u b s t a n c e was o b t a i n e d . T h e n 2 - p r o p a n o l was a d d e d slowly, while i n i t i a l l y s h a k i n g v e r y g e n t l y a n d m o r e v i g o r o u s l y a t t h e end, u n t i l a d a r k r e d d i s h - b r o w n n e t w o r k was o b t a i n e d . T h i s s u b s t a n c e was r e m o v e d f r o m t h e s o l u t i o n b y m e a n s of forceps w i t h b e n t a r m s , t r a n s f e r r e d i n t o a p e t r i d i s h w i t h 75~o e t h a n o l - 0.1 M NaC1, k e p t t h e r e for several m i n u t e s a n d m o v e d in t h e s o l u t i o n f r o m t i m e t o time. T h e s u b s t a n c e was f i n a l l y aird r i e d o n filter p a p e r o v e r n i g h t , a n d k e p t in a sealed vessel a t r o o m t e m p e r a t u r e for i week. (The t i m e s p a n of s t o r a g e is n o t critical for s e v e r a l m o n t h s . ) I m m e d i a t e l y p r i o r t o t h e f i n a l D N A e x t r a c t i o n t h e s u b s t a n c e was allowed t o swell in w a t e r for a p p r o x i m a t e l y 20 m i n a n d was dispersed in a Dounce homogenizer. B o t h t h e i s o l a t e d nuclei as well as t h e s a m p l e j u s t d e s c r i b e d were s u b j e c t e d t o 1 h t r e a t m e n t w i t h p r o n a s e (Merck), CaC1 v a n d LiC1 (final c o n c e n t r a t i o n s 0.25 m g / m l , 5 mM, a n d 1 M , r e s p e c t i v e l y ; in a f i n a l v o l u m e of 10 ml). A f t e r a d d i t i o n of 0.5 m l of a 1 0 % s o d i u m d o d e c y l s u l f a t e solution, a n o t h e r h o u r of i n c u b a t i o n a t 37~ was perm i t t e d . D N A was e x t r a c t e d e s s e n t i a l l y as d e s c r i b e d b y KIRBY z, a p p l y i n g 3 successive e x t r a c t i o n s w i t h e q u a l v o l u m e s of p h e n o l - c r e s o l - h y d r o x y q u i n o l i n e (100 g p h e n o l + 14 m l m-cresol + 0.1 g 8 - h y d r o x y q u i n o l i n e + 25 m l i-I~O) and c h l o r o f o r m . From t h e aqueous phase, DNA was
precipitated with 2-propanol and redissolved in 15 mM NaCl - 1.5 mM Na-citrate pH 7.0. Both DNA preparations were treated subsequently with ribonuclease (RNase A, Boehringer, final concentrations 7.5 Kunitz U/ml; RNase T i, Boehringer, 23 Egami U/ml) for 2 h. Pronase-SDS-, and phenol treatments were performed as described above. The final DNA yields were 0.17 mg (from isolated nuclei) and 0.4 mg (from the direct precipitate). Both DNA samples were scanned in a Beckman DB spectrophotometer with recording attachment. As can be inferred from the Figure, both preparations have almost identical absorption curves. The 260/280 wavelength ratios were 1.85 and 1.86 respectively.
J
300
280
260
240
220
wavetength Into] Absorption curves of DNA extracted from isolated nuclei (solid line) and from a propanol precipitate of Triton-lysed erythrocytes (broken line) derived from a specimen of Salmo irideus. The DNA was solved in 15 mM NaC1- 1.5 mM Na-citrate.
z Acknowledgment. The skilful technical assistance by Miss B. KUNz is gratefully acknowledged. 2 K. S. KIRBY,in Methods o/ Enzymology (Academic Press, New York and London 1968), vol. 12, p. 87.
Specialia
EXPERIENTIA 32/3
A DNA Extraction Procedure Practicable Under Field Work Conditions
j.
SCHMIDTKE 1
Insr /i~r Humc~ngenetik und A nthropologie, Albertstrasse 77, D-78 Freiburg i. Br. (German Federal Republic, B RD), 3 October 1975. Summary. A D N A e x t r a c t i o n p r o c e d u r e is d e s c r i b e d w h i c h is d e s i g n e d t o b e used in field work. A c r u d e i n i t i a l cell p r e c i p i t a t e , w h i c h is easily o b t a i n e d , m a y e n d u r e s t o r a g e of m o n t h s . A p r o c e d u r e is d e s c r i b e d w h i c h allows one t o p r e p a r e D N A f r o m b l o o d of v e r t e b r a t e s w i t h n u c l e a t e d e r y t h r o cytes, a n d w h i c h , i n t h e i n i t i a l stages, r e q u i r e s o n l y t h e simplest technical equipment. The method has been d e v e l o p e d for field w o r k purposes, a n d h a s t h u s f a r b e e n a p p l i e d t o a large n u m b e r b o t h of f r e s h w a t e r a n d seaw a t e r t e l e o s t e a n fish, a n d to a v a r i e t y of a m p h i b i a n s . T h e p r o c e d u r e is e x p l a i n e d h e r e w i t h b l o o d of t h e r a i n b o w t r o u t (Salmo irideus) as a n e x a m p l e . As a c o n t r o l , a c o n v e n t i o n a l D N A e x t r a c t i o n f r o m nuclei isolated f r o m e r y t h r o c y t e s d e r i v e d f r o m t h e s a m e a n i m a l was performed. Methods. 10 d r o p s of b l o o d f r o m t h e c a u d a l vessels were collected in 10 m l 0.1% h e p a r i n - 0.55% NaC1 a q u e o u s s o l u t i o n ( a b o u t 105 cells/ram3). T h i s s u s p e n s i o n was h a l v e d , a n d one h a l f p r o c e s s e d i n t o nuclei. F o r t h i s p u r p o s e t h e e r y t h r o c y t e s were i s o l a t e d b y c e n t r i f u g a t i o n (10 rain, 200 • s u s p e n d e d in 20 m l of R i n g e r ' s A m p h i b -
E .800
!
%
$
t
.600
/
.400
~L
t~
-j
.200
t a n s o l u t i o n c o n t a i n i n g 0.5% glucose, a n d t h e n lysed with Triton-X-100 (Serva; final concentration 0.15%); a n o t h e r 20 m l of R i n g e r ' s a m p h i b i a n - g l u c o s e s o l u t i o n was i m m e d i a t e l y a d d e d to t h e h e m o l y s a t e , f r o m w h i c h nuclei were s p u n d o w n a t 3000 • i n a S o r v a l l centrifuge. T h e second h a l f of t h e o r i g i n a l b l o o d s u s p e n s i o n was p o u r e d i n t o a 100 m l E r l e n m e y e r flask a n d t r e a t e d as follows: A 4~o T r i t o n - X s o l u t i o n was a d d e d u n d e r m o d e r a t e s h a k i n g u n t i l a g e l a t i n e o u s s u b s t a n c e was o b t a i n e d . T h e n 2 - p r o p a n o l was a d d e d slowly, while i n i t i a l l y s h a k i n g v e r y g e n t l y a n d m o r e v i g o r o u s l y a t t h e end, u n t i l a d a r k r e d d i s h - b r o w n n e t w o r k was o b t a i n e d . T h i s s u b s t a n c e was r e m o v e d f r o m t h e s o l u t i o n b y m e a n s of forceps w i t h b e n t a r m s , t r a n s f e r r e d i n t o a p e t r i d i s h w i t h 75~o e t h a n o l - 0.1 M NaC1, k e p t t h e r e for several m i n u t e s a n d m o v e d in t h e s o l u t i o n f r o m t i m e t o time. T h e s u b s t a n c e was f i n a l l y aird r i e d o n filter p a p e r o v e r n i g h t , a n d k e p t in a sealed vessel a t r o o m t e m p e r a t u r e for i week. (The t i m e s p a n of s t o r a g e is n o t critical for s e v e r a l m o n t h s . ) I m m e d i a t e l y p r i o r t o t h e f i n a l D N A e x t r a c t i o n t h e s u b s t a n c e was allowed t o swell in w a t e r for a p p r o x i m a t e l y 20 m i n a n d was dispersed in a Dounce homogenizer. B o t h t h e i s o l a t e d nuclei as well as t h e s a m p l e j u s t d e s c r i b e d were s u b j e c t e d t o 1 h t r e a t m e n t w i t h p r o n a s e (Merck), CaC1 v a n d LiC1 (final c o n c e n t r a t i o n s 0.25 m g / m l , 5 mM, a n d 1 M , r e s p e c t i v e l y ; in a f i n a l v o l u m e of 10 ml). A f t e r a d d i t i o n of 0.5 m l of a 1 0 % s o d i u m d o d e c y l s u l f a t e solution, a n o t h e r h o u r of i n c u b a t i o n a t 37~ was perm i t t e d . D N A was e x t r a c t e d e s s e n t i a l l y as d e s c r i b e d b y KIRBY z, a p p l y i n g 3 successive e x t r a c t i o n s w i t h e q u a l v o l u m e s of p h e n o l - c r e s o l - h y d r o x y q u i n o l i n e (100 g p h e n o l + 14 m l m-cresol + 0.1 g 8 - h y d r o x y q u i n o l i n e + 25 m l i-I~O) and c h l o r o f o r m . From t h e aqueous phase, DNA was
precipitated with 2-propanol and redissolved in 15 mM NaCl - 1.5 mM Na-citrate pH 7.0. Both DNA preparations were treated subsequently with ribonuclease (RNase A, Boehringer, final concentrations 7.5 Kunitz U/ml; RNase T i, Boehringer, 23 Egami U/ml) for 2 h. Pronase-SDS-, and phenol treatments were performed as described above. The final DNA yields were 0.17 mg (from isolated nuclei) and 0.4 mg (from the direct precipitate). Both DNA samples were scanned in a Beckman DB spectrophotometer with recording attachment. As can be inferred from the Figure, both preparations have almost identical absorption curves. The 260/280 wavelength ratios were 1.85 and 1.86 respectively.
J
300
280
260
240
220
wavetength Into] Absorption curves of DNA extracted from isolated nuclei (solid line) and from a propanol precipitate of Triton-lysed erythrocytes (broken line) derived from a specimen of Salmo irideus. The DNA was solved in 15 mM NaC1- 1.5 mM Na-citrate.
z Acknowledgment. The skilful technical assistance by Miss B. KUNz is gratefully acknowledged. 2 K. S. KIRBY,in Methods o/ Enzymology (Academic Press, New York and London 1968), vol. 12, p. 87.
