Massimo Barberis
Histopathology and Molecular Diagnostics Unit

10 - 8- 2011
20 – 8 - 2012
Egfr 1517
K-ras 1387
B-raf 392
C-kit 453
Altri 399
4148 mutational analyses performed for patients seeking for biological therapy at EIO
4148
392 B-RAF suitable for
311 cases regardindg pts with metastatic melanoma
9 Unsatisfactory specimens unsuitable for diagmosis
2.9 %
8 cases in 2011
1 case in 2012

Critical phases for critical specimens
1) Tumor cell / stromal and inflammatory cells
2) Quality of tumor cells
3) Type of nucleic acid extraction
4) Primer selection
Extraction
Nucleic acid must be as pure as possible.
Remove inhibitors, competing NA
Evaluate extraction and purification systems for yield efficency and consistency

Organic DNA extraction method
Cells lysed with a detergent and mixed with phenol/chloroform
Ultrahigh purity of the product
Warning : salt concentration and pH dangerous organic solvents time consuming
Solid phase extraction methods
Silica base or fiber glass based technology
( chaotropic substances such as guanidine hydrochloride)
FFPE Qiagen extraction kit
Automated nucleic acid extraction

Primers design
Let’s revise the lesson…
For a good hybridization :
At least 18-28 nucleotides in lenght
Match Tm of the primers
Keep C-G content between 50-60%
Design primers that are not complementary
Avoid palindromic sequences (to avoid secondary structures)
Avoid all known SNPs ( check data bases *)
Consider the most conserved region of the sequence of interest genome-www2.stanford.edu/cgi-bin/SGD/web-primer frodo.wi.mit.edu/cgi-bin/primer3

Problems that are encountered in “difficult cases”:
Lack of PCR product
Low yield of the desired product
Presence of nonspecific background bands due to mispriming or misextension of the primers
Formation of primer-dimer that competes for amplification with the desired product

Quality and quantity of DNA template
Spectrophotometry, Fluorometry, Electrophoresis
Blood : PCR products between 100-200 bp require 2-4 ng
FFPE : 20-200 ng
Try even with 5-10 ng

Take into consideration and optimize the following
PCR parameters , if necessary
1) Amount of starting template
2) Primer concentration
3) Type of enzyme and enzyme concentration
4) Use modified thermostable DNA polymerases that provide automatic hot-start allowing template DNA and primers to be mixed together and held at a temperature above the threshold of non specific binding of primer to template before amplification
5) Magnesium ion concentration
6) Buffer composition and pH
7) Annealing temperature and number of cycles

A special recipe for a hopeless case….
Various addition or cosolvents such as dimethyl sulfoxide (DMSO), glycerol,
Bovine Serum Albumin can improve amplification:
 Increase the specificity
< Decrease the amount of non specific products interfering with sequencing an old hen makes good broth
McPherson MJ et . PCR Basics from Background to Bench. Springer Verlag, 2000
Innis MA et al. PCR Application. Protocols for Functional Genomics. Academic pPress
1999

The first amplification failed, but we tried again and we got a result in sequencing …..
?? Is the result clinically reasonable or may be attributable to specimen related problems , or to the platform or to the software ??
Re-evaluate the agarose or acrylamide gel, the quality score and the software indicators
The quality of the sequence determined for the wild type control may be useful as a comparison in identifying problems

Troubleshooting low-quality sequencing can be complex and may involve investigating multiple possible causes
Poor template ( low concentration, poor preparation )
Inadequate purification of template prior to sequencing
Poor primer and template annealing
Contaminating sequences in reaction mix
Low Tm, primer-dimer formation
Reagents not performing as expected
And many others

SAMPLE
IDENTIFICATION
(PATHOLOGIST)
TUMORAL CELL
THRESHOLD:
MACRODISSECTION
DNA EXTRACTION:
CONTROL OF DNA
YELD AND QUALITY
PCR: PRIMER
DESIGN AND
AMPLIFICATION
CONDITIONS
PURIFICATION
OF SEQUENCING
PRODUCTS
CYCLE SEQUENCING PCR PRODUCTS
CLEAN UP
(EXOSAP)
GEL ELECTROPHORESIS
CAPILLARY
ELECTROPHORESIS
DATA ANALYSIS

PCR Product
Removal of PCR reaction “residuals”
Primers to avoid multiple sequence dNTPs
Salt to avoid inhibition of sequencing enzyme
Purified Template

Purification Methods of PCR Products
Exo I/ SAP Column/beads Diluition Gel Purification
Sequencing Reaction
Our Experience: we compared columns vs Exo-Sap and we’ve choosen
Exo-sap becouse is: fast and easy cheap high throughput in microplate format
Disadvantage: does not eliminate non specific PCR products

Sequencing Reaction Purification Methods
Ethanol precipitation
Gel Filtration
Columns
BigDye XTerminator kit
Magnetic Beads
Sample Preparation for Injection
Our Experience:
We’re using BigDye Xterminator after comparison with Colums with Ethanol purification.
BigDye Xterminator:
• Simplifies workflow:
• flexible in throughput and makes formamide needless
• No beads removal prior injection
•Removes “dye blobs” efficiently
•No danger to loose pellet (as ethanol)
•Increase signal intensity

To selectively amplify low levels of variant alleles
COLD PCR consists of a particular cycling protocol in which a denaturation step at a lower temperature is applied

The problem of pigmentation
Deparaffized, rehydrated sections
0.5% of potassium permanganate solution for 60’
Washing with distilled water
1% ossalic acid for 1’
Wash three times with distilled water
Dissect the sections and insert the sample in a tube
Proceed to extraction
AFIP red book :
< ossalic acid and potassium permanganate concentration
> Incubation time

11 cases of metastatic heavy pigmented poorly preserved samples of metastatic MM depigmented pigmented
BRAF ex 15
Amplification (Sanger)
Valuable sequence result in 10/11
Cobas
7/11
Valuable sequence result in 7/11
PyroMarkQ96
9/11

Molecular Diagnostics Unit