Tcnicas de Investigacin en Cncer

Departamento de Bioqumica y Gentica

Unidad de Biologa de Tumores Cerebrales
http://www.unav.es/departamento/tumores-cerebrales/

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

Loss of tumor supressors

Homozygous LOH+Mutation LOH+ promoter

Deletion
Methylation

Promoter
Methylation

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

CGH
Tumor
Genomic DNA
Nick Translation

Fluorenscein
Green

Normal
TEXAS
Red

Mix
Hybridization
Washing and
Mounting

Normal
metaphase

Capture Images, Karyotype

and Analysis the results

Nick translation
(traslado de la mella)
- DNAsa: nick
- DNA pol I: polim.
- dNTP fluorescente.
- Longitud de la sonda: proporcion de
enzimas.
- Stop de reaccin: desnaturalizacin y
EDTA

Random priming labelling technique

(al azar)
- primer annealing.
- DNA polimerization by Klenow.
- labeled dNTP.

Ch3
Ch3

Ch11

Ch5
Ch11
Ch7

Ch7

Ch5

Figure 7: CGH Metaphase

of GB30 detected by
Comparative
Genomic
Hybridization.
Chromosome 3 has a gain at
the telomeric region of the q
arm (green color), whereas it
has loss along the rest of the
chromosome (red color).
Chromosome 5 has a loss at
the telomeric region of the q
arm (red color), whereas it
has a gain on rest of the
chromosome (green color).
Chromosome 7 has a high
level of amplification (green
color) throughout the whole
chromosome,
whereas
7p11.2-p12
(EGFR)
demonstrates extra green
color. Chromosome 11 is
normal with yellow color.

Figure 8: CGH Karyotype of GB30

Figure 9: Mean red-to-green ratio profile from pter to qter, obtained from CGH analysis of GB30. The line in the middle of the
profile indicates the base line ratio (1.0), the left and right lines indicate ratio values of 0.85 and 1.17 respectively. The profile illustrates
losses at 3q28-pter, 5q31-qter, 10, 21, gains at 3q26-qter, 5q31-pter, 8, 9, 20 and high level amplification at 2p22-pter and 7.

Figure 6: Summary of all DNA copy number changes detected by CGH in 22 glioblastomas. Each line illustrates
the affected chromosome region in a single tumor sample. The vertical lines on the right side of the chromosome
ideograms indicate gains and the lines on the left represent losses. High-level amplifications are indicated as bold lines.

CGH-array
- metafases NO: representacin del genoma sobre
el array (clones BAC)
- mayor sensibilidad.

FISH en metafase

FISH en interfase

dual-color probe break-apart FISH assay for

FISH studies of fusion genes.

* Mapping of chromosomal
breakpoints
* Detection of subtle translocations
* Identification of marker
chromosomes, homogeneously
staining regions, and double minute
chromosomes,
* Characterization of complex
rearrangements.

SKY-FISH/M-FISH

SKY-FISH/M-FISH
PAINTING PROBES
1. Flow-sorted chromosomes
2. Chromosome-specific probe pools (chromosome painting
probes)
3. Amplified and fluorescently labeled by DOP-PCR
(DOP-PCR: degenerate oligonucleotide-primed PCR)
DIFFERENCES
Different methods for detecting and discriminating the different
combinations of fluorescence after in situ hybridization.

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

PCR DIFERENCIAL

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

Deleciones homocigticas
PCR diferencial
Oligonucletidos problema

PCR

Normal

HD?

Oligonucletidos control

HD

Gen problema
Control interno

Gen prob (t)

Cuantificacin densitomtrica

Control (t)

< 1/3

Gen prob (s)

Control (s)

Marker

G25

G28

G30

G33

300bp
200bp

PTEN-Exon2
GAPDH

100bp

Fig. 1 PTEN homozygous deletion demonstrated in one glioblastoma (G30). Ethidium bromide-stained 2% agarose gel after a
differential PCR assay to detect PTEN homozygous deletion. DNA extracted from 4 glioblastomas show two bands each: the upper one
corresponds to a 202bp fragment of the PTEN exon2 gene and the lower band corresponds to a 160bp fragment of GAPDH. A
homozygous deletion at PTEN exon 2 was scored if the GAPDH band representing the normal DNA was threefold. The 100 base pair
ladder (Pharmacia Biotech) is included (first lane) for size determination.

Marker G19 G27 G28 G30 G33 Normal

300bp
200bp

p16
9q-STS

100bp

Fig. 5: p16 homozygous deletion demonstrated in two glioblastomas (G30 and G33). Ethidium bromide-stained 2% agarose gel after a
differential PCR assay to detect p16 homozygous deletion. DNA extracted from 5 glioblastomas and leukocyte normal DNA show two bands
each: the upper one corresponds to a 235-bp fragment of the p16 exon 2 gene and the lower band corresponds to a 180-bp fragment of a
9qSTS. A homozygous deletion at p16 exon 2 was scored if the STS band representing the normal DNA was threefold. The 100 base pair
ladder (Pharmacia Biotech) is included (first lane) for size determination.

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

Polimorfismos
Variacin del DNA
Mltiples formas de un gen: 2 ms
Mltiples formas de una secuencia de DNA: 2 ms
Frecuencia mayor de un 1% a nivel poblacional
Cualquier regin del genoma: gnica, extragnica, splicing
Se produce la misma o diferente protena
Son causa de diferencias en:
- desarrollo de enfermedades
- respuesta a frmacos, productos qumicos...
- respuesta a microorganismos

Polimorfismos
RFLP (Restriction Fragment Length Polymorphism)
VNTR (Variable Number of Tandem Repeat):
Minisatlites
repeticiones: 11-16 bp
total: 16 Kb

Microsatlites, STR (short tandem repeats)

repeticiones: 1-6 bp.
total: 100-200 bp

SNP (Single Nucleotide Polymorphism)

LOH

Informatividad?

LOH
het

hemi

NO LOH
het

PCR-RFLP-mutaciones
Mutation site

Caso particular, poco utilizado ya

LOH determinada mediante Southern blot
utilizando sondas:
RFLP: Restriction Fragment Length Polymorphism
VNTR: Variable Number of Tandem Repeats

Southern-RFLP-LOH

Southern-RFLP-mutaciones
Mutation site

Southern-RFLP-mutaciones
Mutation site

Southern-RFLP-mutaciones
Mutation site

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

G5

ssDNA

G6

G7

G8 T98G G11 G12 G27

Mutation band

dsDNA

Fig. 2: PCR-SSCP non-isotopic mutation analysis at PTEN exon 2 in DNAs extracted from 7
glioblastomas and one glioblastoma cell line (T98G) that was selected as a positive control.
DNA from T98G cell line contains a point mutation at codon 42, exon 2 of the PTEN gene, where
CTT changes to CGT, and then Leu changes to Arg in the protein sequence. Silver stained 10% nondenaturing polyacrylamide (19:1 acrylamide:bis-acrylamide) gel. ssDNA (single stranded DNA);
dsDNA (double stranded DNA).

Allele Specific PCR (AS-PCR)

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores

Hipermetilacin de promotores

Dinucletidos 5 CpG 3
Del 60 al 90% de los dinucletidos CpG presentan
metilacin a nivel de la citosina.
Islas CpG protegidas frente a la metilacin. Suponen
un 1% del genoma.
- su proporcin de C+G es como mnimo de un 55%.
- su tamao oscila entre 0,5 y 4 Kb.
- no suele presentar metilacin en los promotores
(s en los exones).

Hipermetilacin del promotor

DNA no metilado
metilado

5-TACGGACTGG..............TCGAACCGT-3

DNA
5-TACGGACTGG..............TCGAACCGT-3

Bisulfito sdico
5-TAUGGAUTGG............. TUGAAUUGT-3

Bisulfito sdico
5-TACGGAUTGG..........TCGAAUCGT-3

Oligonucletidos para DNA

no metilado (U)

5-TAUGGAUTGG............TUGAAUUGT-3
AACTTAACA

MSP

Oligonucletidos para DNA

metilado (M)

5-TACGGAUTGG............TCGAAUCGT-3
AGCTTAGCA

MSP
U

Normal Sample
Marker U
M

G6

T24

NC

200bp
100bp

Fig.6: p16 inactivation through promoter hypermethylation in one glioblastoma (G6). Ethidium bromide-stained 2% agarose gel
after a PCR assay to detect unmethylated promoter (U) or methylated promoter (M) regions of the p16 gene. Normal Sample: DNA from
leukocytes, used as a positivecontrol for the unmethylated p16 promoter. The T24 cell line DNA was used as a positive control of p16
methylation. NC: non-DNA PCR, used as a negative control of the PCR reaction. The 100 base pair ladder (Pharmacia Biotech) is
included (first lane) for size determination.

MATERIAL AND METHODS

Bisulfite sequencing
Unmethylated DNA

Methylated DNA

5ATATTA..CGTCGCG..TTATA3

5ATATTA..CGTCGCG..TTATA3

Sodium bisulfite

Sodium bisulfite

5ATATTA...UGTUGUG..TTATA3
AATAT5
5ATATTA
3TATAAT..ACAACAC..AATAT5
ATATTATGTTGTGTTATA
TATAATACAACACAATAT

5ATATTA..CGTCGCG..TTATA3
AATAT5
PCR

5ATATTA
3TATAAT..GCAGCGC..AATAT5
ATATTACGTCGCGTTATA

ATATTATGTTGTGTTATA
TATAATACAACACAATAT

ATATTACGTCGCGTTATA

TATAATGCAGCGCAATAT

TATAATGCAGCGCAATAT

ATATTATGTTGTGTTATA
ATATTACGTCGCGTTATA

TATAATACAACACAATAT

-PCR product sequencing

-Cloning and sequencing

TATAATGCAGCGCAATAT

MATERIAL AND METHODS

Real time methylation assay

Unmethylated DNA
5AAATTA..CGTCGCG..TTATA3

Methylated DNA
5AAATTA..CGTCGCG..TTATA3

Sodium
bisulfite

Sodium bisulfite

5AAATTA...UGTUGUG..TTATA3
AATAT5

5AAATTA..CGTCGCG..TTATA3
AATAT5

5ATATTA
3TTTAAT..ACAACAC..AATAT5

5ATATTA
3TTTAAT..GCAGCGC..AATAT5
U

MATERIAL AND METHODS

Real time methylation

UNMETHYLATED sample:

U
M

T98G cell line, BLU gene

Red: unmethylated DNA

Green: methylated DNA
Brown: sample

METHYLATED sample

GOS3 cell line, BLU gene

MATERIAL AND METHODS

Real time methylation

METHYLATED + UNMETHYLATED

% OF METHYLATION
U

Sample 33, BLU gene

Red: unmethylated DNA

Green: methylated DNA
Brown: sample

SK-NMC cell line, MGMT gene

MS-SSCA

MS-COBRA: Combined bisulfite restriction analysis

CGH (Hibridacin Genmica Comparada)

PCR diferencial
Amplificacin gnica/Deleciones homocigticas
PCR-RFLP y PCR (microsatlites)
Prdida de heterocigosidad (LOH)
PCR-SSCP
Deteccin de mutaciones puntuales
MSP (PCR especfica de metilacin)
Deteccin de hipermetilacin de promotores