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Prévia do material em texto

Working with nucleic 
acids
Prof.	Javier	Álvarez	
An	Introduction	to	Genetic	Engineering,	3rd	Ed.		
Desmond	S.	T.	Nicholl
Working with nucleic acids
Working with nucleic acids
“In addition to what might be termed infrastructure and equip- 
ment, the running costs of a laboratory need to be taken into 
account as this is likely to be a major part of the expenditure in any 
given year after start-up. Funding for research is a major issue for 
anyone embarking on a career in science, and the ability to attract 
significant research funds is a major part of the whole process of 
science. A mid-sized research team in a university (say three 
academic staff, five postdoctoral research assistants, six PhD/MSc 
students, and four technical staff) might easily cost in the region of 
a million pounds a year to run when salary, overhead, equipment, 
and consumables are considered. Thus, a significant part of a 
senior research scientist’s time may be taken up with securing 
grants for various projects, often with no guarantee of available 
long-term funding.”
https://www.jove.com/science-education/10814/dna-isolation
General	steps	in		
DNA	extraction
https://youtu.be/nUfxxIeu1yM
Steps	in	DNA	extraction
LYSIS
PURIFICATION Enzymes
PRECIPITATION	 95%	ethanol
WASH
ELUTION Water	or	
buffer
70%	ethanol
Physical	or	chemical	methods
BUFFER	DE	LISIS	(7.5%	SDS/1.5%	NaCl)
Buffer	(ex.	Tris):	
Maintains	the	pH	of	the	solution			! DNA	remains	stable	
Detergent	(ex.	SDS):	
-	Breaks	plasma	and	nuclear	membranes		! DNA	gets	free	into	
solution	
- Denatures	proteins	! the	proteins	are	accessible	to	the	proteases	
Salt	(ex.	NaCl):	
-		DNA	molecules	won’t	repel	each	other	
-		Facilitates	DNA	precipitation
LYSIS
SDS
Pepsin/Proteinase	K,	etc	
- They	are	proteases	
- Destroy	nuclear	proteins	
- Destroy	citoplasmic	enzymes	that	degrade	DNA	
(DNases)	
- The	treatment	with	protease	increases	the	
amount	of	extracted	DNA	
- They	are	unaffected	by	the	amount	of	detergent	
in	the	lysis	buffer
PURIFICATION
Salts	
-		They	neutralize	charges	on	the	sugar	phosphate	
backbone	
-		Example:	sodium	or	potassium	acetate	
- The	positively	charged	ions	neutralize	the	negative	
charge	of	phosphate	groups	in	the	DNA	
- The	DNA	becomes	less	hydrophilic	=	less	soluble	in	
water
PRECIPITATION	
DNA
* The amount of DNA must be 
large, otherwise it is difficult to 
pellet it.
95%	Ethanol	
-		95%	-	100%	ethanol	
-		Ice	cold.	No	need	to	be	at	-20	or	-70,	it	works	well	at	0	degrees	
-		DNA	is	not	soluble	in	ethanol	
-		Ethanol	has	a	much	lower	dielectric	constant	(24)	than	water	(82),	making	it	much	easier	for	Na+	to	
interact	with	the	PO3-,	shield	its	charge	and	make	the	nucleic	acid	less	hydrophilic,	causing	it	to	“drop	
out”	of	solution	
- When	added	to	a	DNA	solution	in	a	ratio,	by	volume,	of	2:1	in	the	presence	of	0.2	M	salt,	ethanol	
causes	the	nucleic	acids	to	come	out	of	solution		
- DNA	precipitates	and	the	rest	of	cell	contaminants	stay	in	solution	
- Precipitated	DNA	looks	like	spider	threads				! DNA	jellyfish
PRECIPITATION	
DNA
Isopropanol Vs. Ethanol: DNA Solubility
DNA is less soluble in isopropanol so it precipitates faster even at low 
concentrations. The downside however is that the salts will also precipitate in 
isopropanol. With ethanol, the DNA needs to be at a higher concentration to 
flocculate but the salts tends to stay soluble, even at colder temperatures.
DNA precipitates in 35% isopropanol and 0.5 M salt. Using ethanol, the final 
concentration needs to be around 75% with 0.5 M salt. So for the typical 
precipitation protocol, isopropanol is added from between 0.7–1 volumes of 
sample and ethanol is added at 2-2.5 volumes of sample.
PRECIPITATION	
https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/
In Short – Ethanol or Isopropanol?
Use Ethanol If:
1. You have the space to fit two volumes of ethanol to sample in your tube.
2. The sample needs to be stored for a long period of time and will be chilled.
3. You need to precipitate very small DNA fragments.
Use Isopropanol If:
1. Your sample volume is large and you can only fit 1 volume of solvent to your tube.
2. You need large molecular weight species.
3. The DNA concentration in your sample is low.
4. You are in a hurry and want to accelerate the precipitation of nucleic acids at room 
temperature.
PRECIPITATION	
If you use isopropanol, try not to chill it and remember
to wash it thoroughly with 70% ethanol to remove salts.
Ethanol	
-		70%	-	75%	ethanol	
- Eliminates	salts	from	the	pelleted	DNA	
- Eliminates	inhibitors
WASH
-	Rehydration	of	the	pellet	
- Elution	is	done	with	water	of	molecular	grade	(free	of	nucleases)	or	with	
buffer	
- The	amount	of	buffer	determines	the	final	concentration	of	the	DNA	in	the	
solution
ELUTION
To	determine	the	amount	of	extracted	DNA	and	its	
quality	(absence	of	contaminants):		
• Measurements	with	a	spectrophotometer	or	a	
Nanodrop	
• DNA	gel	electrophoresis	
Evaluation	of	the	amount	and	quality	
of	the	DNA
Calidad	del	ADN:	
*Abs	260/280	≥	1.8	
*Abs	260/230	=	2-2.2
DNA	and	RNA	absorb	UV	light	peaking	at	260	
nm	in	aqueous	solutions
Useful	for	the	localization	and	isolation	of	
DNA	and	RNA.
DNA	concentration	and	purity
• An	A260	of	1.0	is	equivalent	to	a	concentration	of	50	︎g	
ml-1	for	double-stranded	DNA,	or	40	 ︎g	ml-1	for	single-	
stranded	DNA	or	RNA.		
• If	the	A280	is	also	determined,	the	A260/A280	ratio	
indicates	if	there	are	contaminants	present,	such	as	
residual	phenol	or	protein.	The	A260/A280	ratio	should	
be	around	1.8	for	pure	DNA	and	2.0	for	pure	RNA	
preparations.
“Programa	de	control	de	calidad	de	ácidos	nucleicos.	Banco	Nacional	
de	ADN	Carlos	III	(Universidad	de	Salamanca).	www.bancoadn.org”
Assessment of nucleic acid purity. Nanodrop Technical note 52646.
DNA	concentration	and	purity
• The	DNA	concentration	can	also	be	determined	by	the	
fluorescence	of	bound	ethidium	bromide.		
• If	the	A280	is	also	determined,	the	A260/A280	ratio	
indicates	if	there	are	contaminants	present,	such	as	
residual	phenol	or	protein.	The	A260/A280	ratio	should	
be	around	1.8	for	pure	DNA	and	2.0	for	pure	RNA	
preparations.
Examples	of	DNA	gels	and	what	they	tell	us
DOI: 10.1186/1746-4811-6-3
Genomic DNA usually 
appears as a high MW 
band at the top.
DOI: 10.1172/JCI2261
DNA degradation 
looks like a smear 
along the lane.
DOI: 10.1093/nar/gkp679
RNA appears at the 
bottom.
Fig. 1. Agarose gel electrophoresis of 
total cellular RNA (100 µg/ml) treated 
with carbon nanotubes in 15 mM 
phosphate buffer (pH 7.0). L, DNA 
ladder; 1, RNA (control); 2–5, RNA with 
carbon nanotubes (38, 75, 150, 300 µg/
ml, sequentially). 
Al añadir más nanotubos de carbono 
estos van atrapando el ARN en 
solución.
Particular	cases
1. Plasmid	DNA	from	bacteria	or	fungi	
2. Genomic	DNA:	
• Bacterial	DNA	
• Fungal	DNA	
• Plant	DNA	
• Animal	DNA
+-
Adams et al. The Biochemistry of the Nucleic Acids (11th ed)https://en.wikipedia.org/wiki/Nick_(DNA)
Use	of	magnetic	beads
Labelling of nucleic acids
• To keep track of how much nucleic material goes to every 
step of a cloning procedure
• (I never had to do it for cloning)
• Radilabelling. Most common isotopes, ordered by the E 
they emit 32P > 35S > 14C & 3H. Portions of each reaction 
may be counted in a scintillation counter
• Fluorescent dyes
• Enzyme-linked labels
Otro: nucleic acid hybridization with a radioactive probe.
Horno de hibridación.
Klenow fragment of DNA pol I
• Because the 5' → 3' exonuclease activity of DNA polymerase I from E. coli makes 
it unsuitable for many applications, the Klenow fragment, which lacks this activity, 
can be very useful in research. 
• The Klenow fragment is extremely useful for research-based tasks such as: 
• Synthesis of double-stranded DNA from single-stranded templates
• Filling in receded 3' ends of DNA fragments to make 5' overhang blunt
• Digesting away protruding 3' overhangs
• Preparation of radioactive DNA probes• The Klenow fragment was also the original enzyme used for greatly amplifying 
segments of DNA in the (PCR) process, before being replaced by thermostable 
enzymes such as Taq polymerase.
Wikipedia
DNA gel electrophoresis
SAGE = Submerged agarose gel electrophoresis
PAGE = Polyacrylamide-based gel electrophoresis 
Agarose vs. polyacrylamide
“Migration in the gel is inversely proportional to the log10 of the number of base pairs.”
Agarose vs. polyacrylamide
“One common technique is SDS-PAGE, in which the detergent SDS (sodium dodecyl sulphate) is 
used to denature multisubunit proteins and cover the protein molecules with negative charges. In 
this way the inherent charge of the protein is masked, and the charge/mass ratio becomes constant. 
Thus, proteins can be separated according to their size in a similar way to DNA molecules.”

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