Nucleótidos y

haptenos para
marcación de ácidos
nucleicos
Dr. Marcelo Cortez
DNA Detection Using Enzymatic Incorporation of Labelled
Precursors
 Use of Radioactive Atoms for Labelling DNA

Despite its many shortcomings the use of radioisotopes for labelling

DNA and RNA is still common practice. The most popular
radioisotopes are 32P (1.71MeV) which gives the highest sensitivity
down to 10 fg and 35S (0.167MeV) which is very popular in DNA
sequencing. Somewhere in between is 33P a new radioisotope with
an energy emission of 0.249 MeV and a half live of 25 days.
NUCLEÓTIDO RADIACTIVO

*
 NUCLEÓTIDO RADIACTIVO

*
 Non Radioactive Labelling of DNA
1. Fluorescein conjugated nucleotide(s)

2. Biotinylated nucleotides

3. Digoxigenin labelled nucleotides

1. Fluorescein conjugated nucleotide(s)

In this case the side group is too bulky for polymerases to

be recognized and can hence not be used for above
reactions. This molecule is nevertheless now widely used
in primers or ddNTPs in connection with automated DNA
sequencing procedures.
 Fluorescence in situ hybridization (2005) of
fluorescein isothiocyanate-labeled hobo DNA and
Fluorescein-12-dUTP, Cy3-labeled mdg1 DNA on the salivary gland
polytene chromosomes from y cn bw sp strain.
Black arrows show chromosome arms, gray
arrows show mdg1 hybridization sites.
2. Biotinylated nucleotides

These modified nucleotides carry a Biotin-residue linked to

dUTP at the 5 'position; this structure mimics well the
conformation of dTTP which has a methyl group at C5 and is
hence accepted by DNA polymerases during chain elongation.
The detection of Biotin-dUTP is based either on a Streptavidin-
conjugate or a anti-biotin MAB-conjugate. These conjugates
consist of Streptavidin or anti-biotin MAB linked to various
“reporter/detector systems" such as calf intestine alkaline
phosphatase or horseradish peroxidase .
Biotin-11-dUTP
 Biotin

Streptavidin is a tetrameric protein (4 x 13kDa) that has various biochemical

applications. The reason for this is the high affinity of the protein to biotin (Ka ~ 1013
M-1). Each monomer of streptavidin binds one molecule of biotin. The binding pocket is
constructed showing three different binding motifs with the ligand as reported in the
structures of the streptavidin-biotin complex by Weber et al., 1989 and Hendrickson et
3. Digoxigenin labelled nucleotides

Digoxigenin is a steroid derived from the plant digitalis

purpurea. This ligand is again joined to the C5-atom of dUTP
via a spacer arm. Despite its relative large size this modified
nucleotide is accepted by DNA-polymerases and built into DNA
during the labelling reactions. The steroid hapten is detected via
a anti-digoxigenin MAB to which one of the above described
AP or HRP dependent reporter functions are linked. With the
new Bio-and Chemiluminescence detection assays available
these non-radioactive systems now have the same sensitivity as
the old radioisotope dependent assays; both however share also
the lack of real time detection (exposure to X-ray film!). By the
way all these detection systems can also be used in labelling
MABs in the detection of antigens.
 DIG

DIG-11-dUTP
For a long time most methods have been restricted to the use of
radiolabelled precursor molecules (ie the NTPs). But with the
emphasis now of transfering rDNA technology into the market
place non-radioactive detection systems become increasingly more
popular. Their main advantage is four-fold:
(i) it allows real time detection (no autoradiography, apart from the
luminescence procedures)

(ii) chemically very stable (no radioactive decay)

(iii)safe to handle; not highly trained personel required for handling

(iv) more accessible to automation

 Calf Intestine Alkaline Phosphatase

Calf intestine alkaline phosphatase (AP) with BCIP

(Bromo-chloro-indolyl-phosphate) and NBT (Nitroblue-
tetrazolium) as substrates and cosubstrates respectively; a
positive signal will give a blue precipitate. Improved AP-
dependent detection systems however are based on either
bioluminescence using D-luciferin-O-phosphate as substrate
(generating D-luciferin which in the presence of firefly
luciferase produces light) or on chemiluminescence via a 1,2-
dioxetane (eg AMPPD) emitting photons directly after the
phosphatase reaction.
 Horse Radish Peroxidase

Horse radish peroxidase (HRP) in the presence of H2O2

and aminoethyl carbazole; more recently HRP and hydrogen
peroxide have been used in a chemiluminescence detection
system using luminol as second substrate giving rise to luminol
radicals which decompose under emission of photons which
can be captured on X-ray film. It is claimed to detect a single
copy gene in as little as 2µg of restricted human genomic
DNA.
 Unión covalente directa de una enzima al DNA
Amersham AlkPhos Direct Labeling and Detection Systems
 Ejercicio
Usted hizo una pasantía en el extranjero donde realizó un experimento analizando la capacidad de la proteína Oct-1 de células HeLa
de unir DNA. En ese laboratorio tenia todo lo necesario para realizar marcaje de ácidos nucleicos con Fosforo 32, obteniendo el
resultado de la izquierda. Llegando a Chile Ud. no tuvo acceso a un laboratorio certificado para trabajo con radioactividad, teniendo
que migrar hacia el marcaje con Digoxigenina, logrando el resultado mostrado a la derecha de la figura.

Unlabeled specific competitor

sequences (where used) were present
at a 200-fold molar excess over
labeled target.

Su profesor tutor no está conforme con el resultado, esperando lograr un experimento con la calidad del P32, sugiriéndole que haga el
marcaje con Biotina.
i) Explique porqué usted cree que su profesor le pidió marcar con biotina, cuales son las ventajas y desventajas por sobre DIG.
ii) Haga una lista de los materiales y reactivos que usted debe reunir para dicho experimento.
iii) Haga un diagrama de flujo de las actividades a realizar.