Call Girls Hyderabad Krisha 9907093804 Independent Escort Service Hyderabad
Reduction of cost of Genetics diagnosis test through FISH & PRINS
1. CEUTEH‘16
Dr.Manish Jain
• Scientist,Department of Reproductive Biology
All India Institute of Medical Sciences
New Delhi
Publication:
• Research Papers (Indexed Journals): 15
• Chapters in Books : 2
• Poster Presentation in National & International
Conferences: 8
• Demostrator cum tutor in National Workshop on
Molecular
• Cytogenetic techniques: 7 times
2. Reduction of cost of Genetics
diagnosis test through FISH & PRINS
Dr. Manish Jain
Scientist
Department of Reproductive Biology
All India Institute of Medical Sciences
New Delhi
3. • A genetic disorder is a genetic problem caused by one or
more abnormalities in the genome, especially a condition that is
present by birth (congenital).
• Defects may occur during gamete formation or fetal
development due to endogenous or exogenous factors.
• These may cause birth defects like developmental delay,
behavioral problems and intellectual disability; abortions or
infertility compromising with reproductive health.
Genetics Disorders
4. GENETIC DEFECTS DURING GAMETE FORMATION
AND FETAL DEVELOPMENT
CHROMOSOMAL
DEFECTS
GENE
DEFECTS
Change in the
structure of
chromosome
Change in
number of
chromosomes
Addition or
deletion of a
nucleotide
Replacement of
one nucleotide
by another
5. SAMPLE COLLECTION
(BLOOD/ AMNIOTIC FLUID/ SOLID TISSUE/ BONE MARROW)
CYTOGENETIC TESTING
KARYOTYPE FISH PRINS aCGH
Methods of Investigations
7. Drawback of Karyotype
• The material doesn't contain metaphase chromosomes
– Unsuccessful cultivation
– It isn't possible to cultivate the tissue from patient
(preimplantation analysis, rapid prenatal examinations,
examinations of solid tumors or autopsy material)
• Analysis of complicated chromosomal rearrangements
• Identification of marker chromosomes
• Analysis of low-frequency mosaic
• Diagnosis of submicroscopic (cryptic) chromosomal
rearrangements
– Microdeletion syndromes
– Amplification of oncogenes and microdeletion of tumor-
suppressor genes in malignancies
8. • A technique that hybridizes a DNA nucleic acid probe to a target
DNA sequence contained within a cell nucleus.
FISH
9. FISH for Detection of Single to Multiple Genetic Events
Single Target
One color
Dual Targets
Two colors
Multiple
Targets
Multi- colors
Allows one to look at multiple genomic changes within a
single cell, without destruction of the cellular morphology.
10. A G G C
T
A
T
T C C G
A
T
A
COVALENT BOND
F
F
Specimen DNA
FISH Probe DNA
• Probe is a nucleic acid (DNA or RNA) that
o labeled with a marker which allows identification and
quantitation
o hybridize to another nucleic acid on the basis of base
complementarity
Probes
12. Company Probe Cost
• Vysis
• Kreatech
• Cytocell
Per slide cost : Rs.8000 – 12000
?
13. Human Probe Preparation
BAC/PAC/Plasmid DNA Probe
cloning
• Clones are shipped as bacterial LB
agar stab culture.
• DH5α E.coli host is harboring a
DNA insert that has been placed
into LB agar.
• Recommended to streak the culture
to single colonies on a LB agar plate
with a specific antibiotic and
prepare a glycerol stock of the
clones for long-term storage at -
80°C.
• Amplification of BAC/PAC/Plasmid
DNA for probe preparation
• Extraction of BAC/PAC/Plasmid
DNA for FISH Probe Preparation
1.a 1.b
1.c 1.d
1.e 1.f
14. Labeling of Probes
Nick translation labeling
• No need for linearization or denaturation of DNA.
• DNA is nicked by DNAse and DNA polymerase I replace excised
nucleotides by labeled/unlabeled nucleotides.
15. Protocol for Direct Nick Translation
• 1.5 ml microcentrifuge tube is placed on ice and to the tube, following ingredients added:
• Sterile distilled H2O Y µl
• 10X conc. nick translation buffer (NTB) 5µl
• [NTB-500 mM Tris-HCL (pH 7.8), 50mM Mg Cl2, 0.5 mg/ml BSA (nuclease-free)
• 100 mM Dithiothreitol 5 µl
• dNTP Nucleotide mixture 4µl
• [dNTP-0.5 mM each of d ATP, dGTP, and d CTP; 0.1 mM dTTP]
• 1 mM fluorochrome –labeled dUTP( DIG/Biotin) 0.5µl
• 1 µg template DNA X µl
• DNase I 5µl
• DNA polymerase I (2 units) 0.5 µl
The ingredients are mixed and tube is centrifuged briefly.
It is incubated at 15°C for 45 minutes.
After 45 mins, 5µl of Diluted DNase is added.
Further incubated at 15°C for 45 minutes.
Immediately after incubation, chilling of the reaction tube is done on ice.
To Stop reaction
• Add
• 0.5 M EDTA 2.5l
• Herring sperm DNA (blocks repetitive sequences) 2.5l
• 3 M sodium acetate (0.1vol) 3.0l
• Cold absolute alcohol 100% (2.5 vol) 500l
34. • It is a method of target DNA sequence detection and localization
that combines features of FISH and polymerase chain reaction
• It is based on in situ annealing of a specific oligonucleotide primer
followed by primer elongation by Taq DNA polymerase in the
presence of labeled nucleotides.
• It is specific, simple, cost effective and rapid technique.
• It does not require a previously labeled probe.
Primer In Situ Labelling
• DNA is linearized and denatured (ssDNA),
• primer anneals and synthesis of new strand of DNA
(incorporating labeled nucleotides) by Taq DNA polymerase
(size should be 200-1000 bp)
PRINS (PRimed INSitu Labeling/Synthesis)
35. PRINS (PRimed INSitu Labeling/Synthesis)
Principle
Metaphase chromosome or Interphase Cell (DNA) onto microscope slide
Oligonucleotide primer (specific for chromosomes) + nucleotides including labeled
+ Taq DNA polymerase + Buffer
Denatured together onto a glass microscopic slide (93C for 3 min)
Annealed at 60C for 10 min
Extension/elongation (with Taq polymerase) at 72C for 15 min
[One (simple) or many (cycling) cycles]
Stop reaction by dipping into EDTA solution (0.2 M) at 65C for 1 min
Washing of excess and unbound probes (room temperature few minutes)
Visualization under fluorescent microscope (for direct PRINS or application of
detection system for indirect PRINS)
36. One primer each specific for centromere/alphoid of chromosome X, Y, 13, 18 & 21 as follows:
Primer Sequence (5’-3’) Concentration (pM) Annealing Temp
X centromere GTTCAGCTCTGTGAGTGAAA 75 68ºC
Yqh TCCATTCGATTCCATTTTTTTCGAGAA 100 56ºC
13 centromere TGATGTGTGTACCCAGCT 100 60ºC
18 centromere ATGTGTGTCCTCAACTAAAG 100 65ºC
21 centromere TGATGTGTGTACCCAGCC 150 61ºC
Primers standardized in our lab
Primed in situ labeling/synthesis
images on metaphase as well as
interphase cells
37. Conclusion
• Lab made probes highly reduce the cost of FISH/PRINS test
making the techniques approachable for patients
• Alphoid, locus specific/Microdeletion syndromes &
Amplification of oncogenes and microdeletion of tumor-
suppressor genes in malignancies can be detected at a much
lower price by using lab made probes as compared to the
commercial probes.
39. Dr. Manish Jain
Scientist
Department of Reproductive Biology
All India Institute of Medical Sciences
New Delhi
Publication:
Research Papers (Indexed Journals): 15
Chapters in Books : 2
Poster Presentation in National & International Conferences: 8
Demostrator cum tutor in National Workshop on Molecular
Cytogenetic techniques: 7 times
Editor's Notes
It affects the functioning of the male and female during all stages of life.
The birth of a healthy baby is the result of fertilization of good quality gametes and development of resulting zygote in mother’s womb.
Genetic investigation includes the analysis of chromosomes, DNA, RNA, genes, and/or gene products to determine whether an alteration is present that is causing or is likely to cause a specific disease or condition.
Genetic defects occur at the level of genes
point mutations,
deletion,
microdeletion
duplication
chromosomes – change in number or structure of chromosomes.
Libraries may have little contamination hence it would be wise, before initiating any experiments, to check 3-5 colonies on LB agar plates. This to make sure that the clone stock is pure.