COLD PCR application used in breast cancer research

COLD-PCR is a modified version of the polymerase chain reaction (PCR) technique used to selectively amplify and enrich rare or minority DNA sequences, such as mutations or genetic variations.

1. PREVALENCE OF CANCER ASSOCIATED GENES IN
BREAST CANCER PATIENTS IN THE HOSPITAL
BASED STUDY FROM SOUTHERN ASSAM
Presented By:-
Jagadish Hansa
Sona Thesis Consultancy

2. Simply being a woman is the main risk factor for
developing breast cancer. Male can also develop.
Two types:- sporadic and hereditary breast cancer.
Hereditary cancer is by BRCA1 and BRCA2 gene.
Commonest cause of death for woman (35-55yr).
Mutations found at the 5’ end increased risk of ovarian
cancer.
BRCA2 mutations confer higher risk of male breast cancer.BRCA2 mutations confer higher risk of male breast cancer.
In Southern Assam this is the 2In Southern Assam this is the 2
ndnd
threat after head and threat after head and
neck cancer in India.neck cancer in India.
introduction

3. The Northeastern region have the highest tobacco related cancer
incidences rate compared to that of rest of the country.
Prevalence of breast cancer in Assam is second highest among all the
states of India.

North-East region of India has different customs, food habits, life-style,
diverse ethnic groups and type and pattern of tobacco use as compared
to the rest of the country.
Statistics itself define that in this southern part of Assam their will be more
requirement of research work.
BREAST CANCER IN NORTH EASTBREAST CANCER IN NORTH EAST

4. Clinical Presentation of Breast
Cancer
Stages of breast cancer
Ductal hyperplasia
Atypical ductal
hyperplasia
Ductal Carcinoma in
situ.
DCIS-MI
Invasive ductal cancer

5. Age
Family History
Reproductive
History
Menstrual
History
Race
Radiation
Treatment with
DES
FA
CTORS FOR breast CANCER
Genetic
Factors

6. B
RCA1
•Localized to the long arm of chromosome 17q21.
•Distributed over 100,000 base pairs of DNA.
•24 exons (22 coding + 2 non-coding) which encode a protein
that is 1,863 aa in length.
•BRCT (C-terminal of BRCA) domain
–Consists of 2 conserved BRCT repeats~90-100 amino
acids long.
•N-terminal – ring-finger domain where Bard1 protein bind.

7. B
RCA2
•Localized to chromosome 13q12.13.
•27 encoding exons distributed over roughly 70 kb of genomic
DNA.
•The exons encode a known sequence of 3418 aa length.
•Carriers also have increased risks of pancreatic, prostate,
laryngeal, and ocular cancers.

8. P53
Localized to chromosome 17p3.1
11 encoding exons distributed over roughly 22 kb of genomic DNA.
The exons encode a known sequence of 393 aa length.
DNA binding region has so many mutations in cancerous patients.

9. B
RCA1 And BRCA2 Mutation Profile
Most of the mutation found in the BRCA1 is in the part of the DNA
binding region, but 185delAG and 5382insC is most frequent mutation
found in breast cancer patients.
In BRCA2, exon 11e and exon 11f have so many mutation.
BRCA1
BRCA2

10. P53 Mut
ation Profile
DNA binding region is mostly effected by the means of
deletion and insertion of nucleotide bases
p53

11. A
ssociation of BRCA1,BRCA2 and p53

12. Pathological Analysis:- The sample diagnosed by
different basis of method were collected from the both centre.
HPE- Histopathology 
refers to the microscopic examination of tissue in
order to study the manifestations of
 clinical sample, histopathology refers
to the examination of a
 biopsy or surgical specimen by a pathologist, after
the specimen has been processed and
 histological section have been
placed onto glass slides.
FNAC- A needle aspiration biopsy is safer and less traumatic than an
open surgical biopsy, and significant complications are usually rare,
depending on the body site.
 
RADIOLOGICAL- Diagnostic radiology is concerned with the use of
various imaging modalities to aid in the diagnosis of disease. Diagnostic
radiology can be further divided into multiple sub-specialty areas.
POST OPERATIVE
CLINICAL DIAGNOSIS

13. Isolation of total genomic DNA and RNA:- The fresh, preserved
paraffin block and the match normal tissue were collected in
eppendorf and the blood (5ml) was collected in EDTA vial for well
process of experiment. The total DNA and RNA was isolated by
standard protocol.
Isolation
(Phenol: Chloroform: Iso-amylalcohol)
Digestion
(TES Buffer, 10% SDS, ProteinaseK)
Incubation
(Over night)
Phenol: chloroform method
Alcohol precipitation
Dissolve
Preservation

14. Good Primer’s Characteristic
Uniqueness: one and only one target site.
Length: the length of primer has to be at least 15 bases to 21 base.
Base Composition: Average (G+C) content around 50-60% will
give us the right melting/annealing temperature for ordinary PCR
reactions, Base composition affects hybridization specificity and
melting/annealing temperature.
Melting Temperature: Tm is characteristics of the DNA
composition; Higher G+C content DNA has a higher Tm due to
more H bonds.
Annealing Temperature: Annealing Temperature, T
anneal
– the
temperature at which primers anneal to the template DNA. It can be
calculated from T
m
.
Absence of Dimerization Capability: Avoid hairpin, self-dimer and
Dimer structure.
T
anneal
= T
m_primer
– 4C

15. Computer-Aided Primer Design
Primer design is an artart when done by human beings, and a
far better done by machinesfar better done by machines.
 

Some primer design programs we use:
- Oligo: Life Science Software, standalone application
- GCG: Accelrys, ICBR maintains the server.
- Primer3: MIT, standalone / web application
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
- BioTools: BioTools, Inc. ICBR distributes the license.
- Others: GeneFisher, Primer!, Web Primer, NBI oligo program, etc.
Melting temperature calculation software:
- BioMath: http://www.promega.com/biomath/calc11.htm

16. C
OLD-PCR
C
o-amplification at low
den
aturation temperature

17. Princi
ple of cold-pcr
A single nucleotide mismatch anywhere along a double-
stranded DNA sequence generates a small but predictable
change to the melting temperature of DNA (Tm) for that
sequence.
Abbreviation used in COLD-PCR
Tm= amplicon temperature
Tc = critical denaturation temperature
Ta = anneling temperature

18. It is used to identify
all possible
mutations in the
targeted DNA
However the
enrichment may be
modest compared to
fast COLD-PCR
It can be used to provide a
strong enrichment of Tm-
reducing mutations, which
comprise the majority
mutations in cancer, and is
simpler and convenient
because the amplification
is completed in less time.

19. RESULTS
Chapter-1
Survey of cancer
effected patients
Collection of tissue
from patients

20. Chapter-1 (SURVEY)
Different cancer incidences in southern
Assam

21. Chapter-1 cont.
Different type of cancer incidences in the
year 2008, 2009 and 2010.

22. Chapter-1 cont.
Breast cancer by Age Group.

23. Collection of sample
Sample ID S/A/R Year District Hospital
AUBC1 F/35/H 2009 CACHAR CCHRC
AUBC2 F/46/H 2009 HKD CCHRC
AUBC3 F/70/H 2009 CACHAR CCHRC
AUBC4 F/56/H 2009 CACHAR SMC
AUBC5 F/67/H 2009 CACHAR CCHRC
AUBC6 F/70/H 2009 KXG SMC
AUBC7 F/76/H 2009 HKD CCHRC
AUBC8 F/65/H 2009 CACHAR CCHRC
AUBC9 F/45/H 2009 HKD CCHRC
AUBC10 F/49/H 2009 HKD SMC
AUBC11 F/56/H 2009 CACHAR SMC
AUBC12 F/64/M 2009 CACHAR SMC
AUBC13 F/70/M 2009 CACHAR CCHRC
AUBC14 F/56/H 2009 KXG CCHRC
AUBC15 F/66/H 2009 KXG SMC
AUBC16 F/65/M 2010 CACHAR CCHRC
AUBC17 F/63/H 2010 KMG CCHRC
AUBC18 F/35/H 2010 HKD CCHRC
AUBC19 F/39/H 2010 HKD CCHRC
AUBC20 F/42/M 2010 HKD SMC
AUBC21 F/68/H 2010 CACHAR SMC
AUBC22 F/43/H 2010 KXG SMC
AUBC23 F/53/H 2010 CACHAR SMC
AUBC24 F/44/H 2010 KXG CCHRC
AUBC25 F/34/H 2010 HKD SMC
AUBC26 F/36/H 2010 HKD SMC
AUBC27 F/45/M 2010 CACHAR SMC
AUBC28 F/80/H 2010 CACHAR SMC
AUBC29 F/69/H 2010 CACHAR SMC
AUBC30 F/56/H 2010 CACHAR SMC
AUBC31 F/57/H 2010 CACHAR CCHRC
AUBC32 F/67/H 2010 CACHAR SMC

24. RESULTS
Chapter-2
Patholological
analysis
Isolation of total
genomic DNA and
RNA

25. Pathological Analysis:- The sample diagnosed
by different basis of method were collected from the both
centre.
Chapter-2

26. Epithelial pearls
Laminated keratin
Microscopic view of the tumor cells indicates columns of epithelial cells
growing down in the dermis. The lower parts of the columns often appear as masses the
same process of cornification goes on as occurs normally on the surface. Granules
appear in the cytoplasm and the cells become converted to hyaline structure masses of
keratin which stain brightly with eosin and are identical with the horny material on the
surface of the skin. Such a general picture is well named as epidermoid carcinoma. The
cornified masses are known as cell nests or epithelial pearls. The outer cells of the pearls
are arranged in a concentric manner as shown in fig.
Microscopic view of the tumor cells
Chapter-2 cont.

27. Chapter-2 cont.
Genomic DNA isolation from samples
1kb BC1 BC2 BC3 BC4
Isolation from tissue
Isolation from Blood
Isolation from FNAC
Paraffin embedded tissue
was isolated using the
Quick Extract FFPE DNA
Extraction Kit from
Epicentre Biotechnologies.

28. RESULTS
Chapter-3
Primer Design
Hotspot mutation
detection

29. p
rimers Properties
Primer Nucleotide LengthATGC
GC
content
Mol.
WeightTm
BRCA1-185delAG-FATT GGA ACA GAA AGA AAT GG 201036135%6247.252.3 C
BRCA1-185delAG-RCCT AGT ATG TAA GGT CAA TTC T 22683536%6724.556.4 C
BRCA1-1014delGT-FACA GCA TGA GAA CAG CAG 18714650%5550.753.8 C
BRCA1-1014delGT-RCAC AGG GGA TCA GCA TTC AGA 21636652%6464.361.2 C
BRCA1-3889delAG-FTCT ACT AGG CAT AGC ACC GTT 21564648%6381.259.5 C
BRCA1-3889delAG-RCTT CCA ATT CAC TGC ACT GTG 21473748%6332.259.5 C
BRCA1-5382insC-FGAA GTC AGA GGA GAT GTG GTC 21649252%6575.461.2 C
BRCA1-5382insC-RTCT TAC AAA ATG AAG CGG C 19744442%5820.953.0 C
BRCA2-6178delT-FTCT ACT AGG CAT AGC ACC GTT 21564648%6381.259.5 C
BRCA2-6178delT-RACA GCA TGA GAA CAG CAG 18714650%5550.753.8 C
p53-E8-F GCT TCT CTT TTC CTA TCC TG 201
1
02745%5975.956.4 C
p53-E8-R CTT ACC TCG CTT AGT GCT 18273650%5416.653.8 C
Chapter-3 (PRIMER DESIGN)

30. Pri
mers used for breast cancer
s
creening
Sequence Name
Primers
Product
size
Tc
BRCA1-185delAG-F ATT GGA ACA GAA AGA AAT GG
185bp85.5
BRCA1-185delAG-R CCT AGT ATG TAA GGT CAA TTC T
BRCA1-1014delGT-F ACA GCA TGA GAA CAG CAG
195bp86
BRCA1-1014delGT-R CAC AGG GGA TCA GCA TTC AGA
BRCA1-3889delAG-F TCT ACT AGG CAT AGC ACC GTT
192bp85.5
BRCA1-3889delAG-R CTT CCA ATT CAC TGC ACT GTG
BRCA1-5382insC-F GAA GTC AGA GGA GAT GTG GTC
168bp82.3
BRCA1-5382insC-R TCT TAC AAA ATG AAG CGG C
BRCA2-6178delT-F TCT ACT AGG CAT AGC ACC GTT
172bp83
BRCA2-6178delT-R ACA GCA TGA GAA CAG CAG
P53-E8-F GCT TCT CTT TTC CTA TCC TG
167bp81
P53-E8-R CTT ACC TCG CTT AGT GCT
Chapter-3 cont.
REFERENCE:- BIC (Breast cancer information core)

31. I
dentification of the t
c
A series of denaturation temperatures lower than the Tm at steps of 0.5 1C
were then applied for COLD-PCR until the temperature was low enough that
no specific PCR product was produced.
We then set the Tc as the lowest temperature that reproducibly yielded a
substantial PCR product. In all cases, to achieve a substantial differential in the
amplification efficiency of wild-type and point mutation–containing sequences,
For each amplicon DNA sequence there is a Tc that is typically 0.2 to 1.5
0
C
lower than the amplicon melting temperature (Tm), below which PCR
efficiency drops abruptly.
•In a PCR reaction, we set the following denaturation temperatures:
(a) A conventional temperature, 98
0
C;
(b) Temperature equal to amplicon Tm;
(c) Temperature equal to amplicon Tm–0.5
0
C;
(d) Temperature equal to amplicon Tm–1.0
0
C;
(e) Temperature equal to amplicon Tm–1.5
0
C.

32. C
ycling condition for
cold-
pcr Amplification
Full P
CR
F
ast PCR
10 cycle
s
25 cycle
s
95
0
C, 15 s;
55
0
C, 30 s;
72
0
C, 1 min;
95
0
C, 15 s;
55
0
C, 30 s;
72
0
C, 1 min;
30 cycle
s
30 cycle
s
95
0
C, 15 s;
70
0
C, 8 min;
83.5
0
C, 3 s;
55
0
C, 30 s;
72
0
C, 1 min.
83.5
0
C, 3 s;
55
0
C, 30 s;
72
0
C, 1 min.
Identify all possible mutationAchieve the highest mutation
enrichment
Total = app. 6hr. Total = app. 2hr.

33. PCR Amplification by
COLD-PCR
Amplification of 192bp DNA
amplicon in 2% agarose gel.
Amplification of 168bp DNA
amplicon in 2% agarose gel.
1kb BC1 BC2 BC3 BC4 BC5
Chapter-3 cont.

34. RESULTS
Chapter-4
DNA Sequencing
Bioinformatical
Analysis

35. DNA PURIFICATION
All PCR products were purified using PCR Purification Kit (Qiagen, UK)
DNA SEQUENCING
The purified PCR product was sequenced both from forward and reverse
side using automated DNA sequencer (ABI3700).
C
OLD PCR Product sequencing
Chapter-4

37. Sequence Analysis by Bioinformatical Tools
Chapter-4 cont.

38. Insertion (G), in p53
Deletion (G), in p53
Chapter-4 cont.

39. Chapter-4 cont.
Deletion (185delAG), in BRCA1

40. Normal sequence having GT 1014delGT sequence
Normal sequence of BRCA1 3889delAG sequence of BRCA1
Peak variation between Conventional and COLD-PCR
Mut
ation detection through
cold-
pcr

41. D
ISCUSSION
Three previously reported deleterious frame-shift mutations resulting in a
premature termination codon were identified in BRCA1: 185delAG in exon
2; 1014delGT and 3889delAG in exon 11 in southern part of the Assam.
Surprisingly, we have found 185delAG in a Northeast Indian, Hindu
patient residing in Cachar district who claimed to have no Jewish ancestry.
BRCA1 1014delGT was detected in a Muslim index case of very early
onset disease [age 24] without any family history. Interestingly, the same
mutation was reported in a heterogeneous Italian population intermixed
with French, German, and Slovenian ethnic groups and a large number of
Muslim immigrants.
The 3889delAG mutation is located towards the C terminus of BRCA1,
within the transcriptional activation domain [39, 40], a region also reported
to interact with the BRCA2 protein, which plays an important role in double
stranded break (DSB) repair.
The above mutations have been reported in the Breast Cancer
Information Core (BIC) website in breast cancer cases of diverse ethnic
origins.

42. Functional significance
Missense mutations may be pathogenic, depending upon the
nature of the amino acid substitution and its effect on protein
structure or function.
 In general, missense alterations in conserved protein motifs
are more likely to be deleterious. Missense amino acid changes
in the p53- binding domain or the transactivation domain of
BRCA1 adjacent to a BRCT repeat have been shown to be
pathogenic.
A possible explanation for the earlier age of disease onset in
BRCA1/2 mutation carriers could be high circulatory estrogen
levels in younger women compared to elderly women.

43. s
ummary
Survey and collection of samples from patients through analyzing the
data from tumor registry office to find the prevalence of breast cancer
of the year 2008, 2009 and 2010 at Cachar Cancer Hospital and Silchar
medical college .
Collection of 32 cancer samples on the basis of cytohistochemistry
report of the pathology laboratory of the hospital.
Detection and analysis of mutation in BRCA1, BRCA2 and P53 gene
from the samples using COLD PCR. From 32 patients 7 had no
mutational changes with respect to the experimental design of the gene.
Mutational study by the help of COLD PCR, which enriches the sequence
chromatogram helps us to find out mutation part of the cancer associated
genes.

44. From this study we found some Sevier change in the cancer
associated genes responsible for breast cancer disease, for this
extensive mutation screening of high-risk breast cancer patients
should take whole genome sequencing with proper genetic
counselling, since female carriers of mutations in these genes are
also at a high risk for developing a second malignancy in the ovary.
Uses of COLD-PCR technique may also potentially be used to
overcome from the mutation site in different genes which causes the
prognosis of cancer, as because the downstream assays after the
COLD-PCR will increase their ability to identify subtle genetic
changes.
For validation of my study more samples analysis will required.
Immunohistochemistry of the cancerous tissue will be carried by
the help of different antibodies.
Epigenetic study will be carried out from the collected cancerous
tissue to check the methylation of the genes.

45. References
1)Durant, S.T. and Nickoloff, J.A. Good timing in the cell cycle for precise
DNA repair by BRCA1. Cell Cycle 2005, 4:1216–1222.
2)Honrado, E., Benítez, J., Palacios, J. The molecular pathology of
hereditary breast cancer: Genetic testing and therapeutic implications.
Mod. Pathol. 2005, 18(10): 1305-1320.
3)Li J, Makrigiorgos GM. COLD-PCR: a new platform for highly improved
mutation detection in cancer and genetic testing. Biochem Soc Trans,
2009, 37:427–432.
4)Mancini,
 I., Santucci, C., Sestini, R., Simi, L., Pratesi, N., Cianchi,
F.,
 Valanzano, R., Pinzani P. and Orlando, C. The Use of COLD-PCR
and High-Resolution Melting Analysis Improves the Limit of Detection of
KRAS 
and 
BRAF 
Mutations in Colorectal Cancer.
The J. Mol. Diagn.,
2010.
5)Rennert, G., Bisland-Naggan, S., Barnett-Griness, O., Bar-Joseph, N.,
Zhang, S., Rennert, H.S., Narod, S.A. Clinical outcomes of breast
cancer in carriers of BRCA1 and BRCA2 mutations. N. Engl. J.
Med.2009, 357: 115-123.
6)www.cancer-genetics.com (BIC)

46. Paper Publication
PUBLISHED PAPER
SK Ghosh, B Choudhury, J Hansa, R Mondal, M Singh, S Duttagupta, A Das, R Kumar, R
S Laskar, R Kannan and P R Ghosh, Human Papillomavirus Testing for Suspected
Cervical Cancer Patients from Southern Assam by Fast-PCR, Asian Pacific Journal of
Cancer Prevention. 2011, 12, pp 749-751.
 
COMMUNICATED PAPER
Jagadish Hansa, Rosy Mondal and Sankar Kumar Ghosh, C-tract of Mitochondrial D-
loop mutation in Cancerous tissue by Co-amplification at lower denaturation
temperature (COLD)-PCR, BMC Cancer, 2011.
 
Ghosh. Sankar, Mondal. Rozy, Hansa. Jagadish and Ghosh. Probal, Fast-PCR Diagnosis
in Female Genital Tuberculosis (FGTB) of the Infertile Women in Southern Assam,
India, Epidemiology and Infection, 2011.
 
Rosy Mondal, Fazlur Rehman, Jagadish Hansa, Ruhina Laskar and Sankar Kumar Ghosh,
Association of mitochondrial D-loop mutation with genetic polymorphism in
glutathione S-transferase GSTM1 and GSTT1 increase susceptibility to oral squamous
cell carcinoma in Northeast Indian populations, Carcinogenesis, 2011.
 
MANUSCRIPT PREPARATION
1)Screening of BRCA1 gene mutations by the help of COLD-PCR from Northeast
India.
2)Prevalence of head and neck cancer in southern Assam.

47. ABSTRACT, ORAL/POSTER PRESENTATION
AT DIFFERENT CONFFERENCE
1)Hansa Jagadish, Mondal Rosy, Kannan Ravi and Ghosh K. Sankar
(2011), Mitochondrial DNA Detection at the D310 Region by COLD-
PCR in Cancerous Tissue, 98
th
ISC, 2011, Chennai.
2)Mondal Rosy, Hansa Jagadish, Kannan Ravi and Ghosh K. Sankar
(2011), Detection of Mitochondrial Genome Variation in Head and Neck
Cancer, 98
th
ISC, 2011, Chennai.
3)Singh M., Hansa J., Choudhury B., Duttagupta S., Das A., Kumar R.,
Laskar S R., Kannan R., Ghosh K S., (2011), PCR Based Diagnosis of
HPV Infection in Suspected Cervical Cancer Patients for Southern
Assam: A Report. 98
th
ISC, 2011, Chennai.
4)Hansa Jagadish, Mondal Rosy, Biswas R. and Ghosh K. Sankar
(2010), PCR Based Disgnosis of HPV from Cancer Patients in the
Hospital Based Study from Southern Assam, 97
th
ISC, 2010,
Tiruvanantapuram.
5)Mondal Rosy, Hansa Jagadish, Kannan Ravi and Ghosh K. Sankar
(2010), Prevalence of Oral Cancer and its Association with
Mitochondrial DNA Mutation from Northeast India, 97
th
ISC, 2010,
Tiruvanantapuram.
SEQUENCE SUBMITTED TO NCBI - 23 sequences (JN603607-JN603629).

48. SEQUENCE SUBMITTED TO NCBI – 23 sequences
BankIt1473753 SGSC JN603607; BankIt1473753 SGIS JN603608
BankIt1473753 SGBS JN603609; BankIt1473753 SGSSD JN603610
BankIt1473753 SGSB JN603611; BankIt1473753 SGPG JN603612
BankIt1473753 SGAB JN603613; BankIt1473753 SGARS JN603614
BankIt1473753 SGNSK JN603615; BankIt1473753 SGAMAA JN603616
BankIt1473753 SGBAB JN603617; BankIt1473753 SGBC JN603618
BankIt1473753 SGMKP JN603619; BankIt1473753 SGMKB JN603620
BankIt1473753 SGSDG JN603621; BankIt1473753 SGMS JN603622
BankIt1473753 SGJS JN603623; BankIt1473753 SGSSM JN603624
BankIt1473753 SGBKT JN603625; BankIt1473753 SGAH JN603626
BankIt1473753 SGMMD JN603627;BankIt1473753 SGRKM JN603628
BankIt1473753 SGPRG JN603629
Going to submit: approx. 60 Going to submit: approx. 60