Molecular Biology analyses

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Molecular Biology analyses

A

Molecular Biology Analyses

Via F. Ferrer 25/27 - 21052 Busto Arsizio (VA) - Italy

Tel +39 0331 652911 - Fax +39 0331 652919 - www.tomalab.com


A1. Molecular screening of β Talassemia

Method 1:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

IVS 2745 - IVS 2.1 - Codon 39 - IVS 1110 - IVS 1.6 - IVS 1.5-IVS

1.1 - Codon 8 / 9 - Codon 8 - Codon 6 - Codon 5 - 87 - 30

– 92 - 101 - Codon 30 -- IVS 1.2 - IVS 1.116 - IVS 1-25- Codon

44 - IVS 2.844

A2. Diagnosis prenatal of Talassemia

Method 1:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

IVS 2745 - IVS 2.1 - Codon 39 - IVS 1110 - IVS 1.6 - IVS 1.5-

IVS 1.1 - Codon 8 / 9 - Codon 8 - Codon 6 - Codon 5 - 87

- 30 – 92 - 101 - Codon 30 -- IVS 1.2 - IVS 1.116 - IVS 1-25

- Codon 44 - IVS 2.844

Method 2:

ARMS (amplification with primers specific for the wild type

and mutated sequence) and data collection on agarose gel

Mutations investigated:

IVS 2745 - IVS 2.1 - Codon 39 - IVS 1110 - IVS 1.6 - IVS 1.1

- IVS 1.2

A3. Molecular screening of sickle cell disease

Method:

gene amplification and mutation detection of by Reverse Dot Blot

Mutation investigated:

haemoglobin S gene

A4. Prenatal diagnosis of sickle cell disease

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations Investigated :

hemoglobin S variant

NOTE (#) page 8

A5. Molecular Screening of haemoglobin C

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

hemoglobin C variant

A6. Prenatal diagnosis of haemoglobin C

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

hemoglobin C variant

NOTE (#) page 8

A7. Molecular screening of Cystic Fibrosis gene

(CFTR)

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

F508 - G542X - N1303K - 1717-1GA - W1282X - G551D

- R553X - S1251N - R560T - 3905insT - Q552X - 1507 -

394delTT - G85E - 621+1GT - R117H - 1078delT - R347P -

R334W - E60X 711+5GA - 2789+5GA - R1162X - 3659delC

- 3849+10kbCT - 2143delT - A455E - 2183AAG - 2184delA

- CFTRdele2,3(21kb) - 711+1GT - 3272-26AG - 1898+1GA -

I148T 3199del6 - 3120+1GA - 1259insA - 4016insT - 4382delA

- 852del22 - D579G - G1244E - G1349D I502T - L1065P -

R1158X - T338I - S549R(AC) - 991del5 - D1152H - 1898+3AG

- R1070Q - R1066H - R347H - 621+3AG - E217G - R334Q

and poly T variant allele located in intron 8 of CFTR

A8. Prenatal diagnosis of Cystic Fibrosis gene

(CFTR)

Method:

gene amplification and mutation by Reverse Dot Blot

Mutations investigated:

F508 - G542X - N1303K - 1717-1GA - W1282X - G551D

- R553X - S1251N - R560T - 3905insT - Q552X - 1507 -

394delTT - G85E - 621+1GT - R117H - 1078delT - R347P -

R334W - E60X 711+5GA - 2789+5GA - R1162X - 3659delC

- 3849+10kbCT - 2143delT - A455E - 2183AAG - 2184delA

- CFTRdele2,3(21kb) - 711+1GT - 3272-26AG - 1898+1GA

- I148T 3199del6 - 3120+1GA - 1259insA - 4016insT -

4382delA - 852del22 - D579G - G1244E - G1349D I502T

- L1065P - R1158X - T338I - S549R(AC) - 991del5 - D1152H

- 1898+3AG - R1070Q - R1066H - R347H - 621+3AG -

E217G - R334Q and poly T variant allele located in intron

8 of CFTR

NOTE (#) page 8

A9. Detection of CFTR exonic rearrangements

associated with Cystic Fibrosis

Method:

multiplex Ligation dependent Probe Amplification (MLPA

P091) and fragment analysis by capillary electrophoresis

Principle:

detection of deletions / duplications of all CFTR exons

A10. Molecular Screening of Spinal Muscular

atrophy Type 1, 2 and 3

Method 1:

real time PCR (quantitative gene amplification) of the SMN1 gene

Method 2:

gene amplification and data fragment analysis by semi-quantitative

capillary electrophoresis of the SMN1 gene

Principle:

detection of the deletion of SMN1 gene (exon 7)

A11. Prenatal diagnosis of Spinal Muscular

atrophy Type 1, 2 and 3

Method 1:

real time PCR (quantitative gene amplification) of the SMN1 gene

Method 2:

gene amplification and fragment analysis by semi quantitative

capillary electrophoresis of the SMN1 gene

Principle:

detection of the deletion of SMN1 gene exon 7

NOTE (#) page 8

A12. Pre-and postnatal diagnosis of uniparental

disomy

Method:

amplification of microsatellite markers located on imprinted

chromosomes (chromosomes 2, 6, 7, 11, 14, 15, 16 and 20) and

fragment analysis by capillary electrophoresis

Principle :

assessment of the parents to proband/fetus segregation of the

imprinted chromosome

NOTE (#) page 8


A13. Rhesus fetal diagnosis on maternal blood

Method:

real time PCR (quantitative gene amplification) of the Rh gene

factor

Principle:

fetal Rh factor typing in maternal blood

A14. Pre-and postnatal diagnosis Paternity Testing

Method:

amplification of microsatellite markers located on different chromosomes

and fragment analysis by capillary electrophoresis

Principle:

assessment of the father to proband chromosome segregation

NOTE (#) page 8

A15. Molecular Detection of SRY gene

A17. Molecular Analysis of Coagulation Factor II

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

G20210

A18. Molecular analysis of factor V Leiden

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

G1691A

A19. Molecular analysis of MTHFR

Method:

gene amplification and mutation detection by Reverse Dot Blot

A22. Detection of trisomies 21, 18, 13 and sex

chromosome aneuploidy by QF-PCR

Method:

gene amplification of microsatellite markers located on chromosomes

13, 18, 21 and X/Y and fragment analysis by capillary

electrophoresis

Principle:

assessment of the dosage of chromosomes 13, 18, 21 and X/Y

NOTE (#) page 8

A23. Pre-and postnatal diagnosis of Charcot-

Marie-Tooth syndrome type 1A (CMT1A)

and Hereditary Neuropathy with liability

to Pressure Palsies (HNPP)

Method 1:

Multiplex Ligation-dependent Probe Amplification (MLPA

P033B) and fragment analysis by capillary electrophoresis

Principle:

detection of deletions / duplications of the subtelomeric

regions of all chromosomes (except p-arm of acrocentric

chromosomes)

A25. Detection on unbalance of genes mapped

on chromosome X in the case with X-linked

mental retardation

Method:

Multiplex Ligation-dependent Probe Amplification (MLPA

P106) and fragment analysis by capillary electrophoresis

Principle:

detection of deletions / duplications of genes mapped to chromosome

X associated with X-linked mental retardation

Investigated Genes:

FMR1-FMR2-ARX-ARHGEF6-RPS6KA3-OPHN1-GDI1-

PQBP1-SLC6A8– FACL4-DCX

Method:

Amplification of the SRY gene and fragment analysis by capillary

electrophoresis

Principle:

to define the presence or absence of the SRY gene, responsible

for the male sex differentiation

NOTE (#) page 8

A16. Microdeletion of Y chromosome (AZF)

Method:

amplification of STS of the AZF region located on the long arm

of chromosome Y and fragment analysis data on agarose gel

Principle:

detection of deletions of the AZF region

Mutations investigated:

C677T - A1298C

A20. Molecular analysis of PAI-1 gene

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

4G/5G

A21. Molecular analysis Hemochromatosis

Method:

gene amplification and mutation detection by Reverse Dot Blot

Mutations investigated:

C282Y - H63D

Method 2:

semi quantitative amplification of microsatellite markers located

into the CMTIA critical region and fragment analysis by

capillary electrophoresis

Principle:

detection of deletions / duplications of the PMP22 locus associated

with CMT1A and HNPP syndromes

NOTE (#) page 8

A24. Detection of Subtelomeric unbalances in

cases with idiopathic mental retardation

Method:

Multiplex Ligation-dependent Probe Amplification (MLPA

P036/P070) and fragment analysis by capillary electrophoresis

A26. Characterization of supernumerary marker

Method 1:

Multiplex Ligation-dependent Probe Amplification (MLPA

P036/P070) and fragment analysis by capillary electrophoresis

Method 2:

Multiplex Ligation-Dependents Probe Amplification (MLPA

P181/P182) and fragment analysis by capillary electrophoresis

Method 3:

Array Comparative Genomic Hybridisation (aCGH) with average

resolution of 0.6Mb

Principle:

detection of subtelomeric and pericentromeric regions (MLPA)

and of the all whole chromosomes (aCGH)

NOTE (#) page 8


A27. Detection of aneuploidy of all chromosomes

Method 1:

Multiplex Ligation-dependent Probe Amplification (MLPA

P036/P070) and fragment analysis by capillary electrophoresis

Method 2:

Multiplex Ligation-dependent Probe Amplification (MLPA

P181/P182) and fragment analysis by capillary electrophoresis

Method 3:

Array Comparative Genomic Hybridisation (aCGH) with average

resolution of 0.6Mb

Principle:

detection of unbalance (deletions / duplications) involving

subtelomeric-pericentromeric regions (MLPA) or the whole

chromosomes (aCGH)

NOTE (#) page 8

A28. Search imbalance of chromosome X in

cases with idiopathic growth retardation

and Leri-Weill Discondrosteosis

Method:

Multiplex Ligation-dependent Probe Amplification (MLPA

P018) and fragment analysis by capillary electrophoresis

Principle:

detection of deletions / duplication of all exons of the SHOX

gene, located to the PAR1 region

NOTE (#) page 8

A29. Assessment of the X chromosome

inactivation

Method:

amplification of the microsatellite marker, digestion with methylation

sensitive restriction enzymes Hha I - Hpa II and located

into the AR gene

Principle:

investigation of the X chromosome inactivation state in females

by capillary electrophoresis

NOTE (#) page 8

A30. Investigation of whole genome unbalances

by array CGH

Method:

Array Comparative Genomic Hybridisation (aCGH) with average

resolution of 0.6Mb

Principle:

Dye-Swap experiment with reverse labelling of the test and of

the reference samples

A31. Molecular analysis of the Fragile X

Method:

PCR and Southern Blot

Principle:

PCR and Southern Blot analysis

DETECTION OF THE NUCLEIC

ACIDS OF THE INFECTIVE AGENTS

A32. Detection of the Toxoplasma genome in

amniotic fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of the Toxoplasma in amniotic fluid.

Analysis of the region B1

A33. Detection of the Epstein Barr virus genome

in amniotic fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of the EBV viral genome in amniotic

fluid

A34. Research of the Parvovirus B19 genome in

amniotic fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of the viral genome in amniotic fluid.

Analysis of the region NS1

A35. Detection of the Rubeo genome in

amniotic fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of the viral genome in amniotic fluid.

Analysis of the region E1

A36. Detection of the CMV genome on amniotic

fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of the viral genome in the amniotic

fluid. Analysis of region IEA1

A37. Detection of the Herpes Zoster virus

genome in amniotic fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of the viral genome in amniotic fluid.

Analysis of the region 28

A38. Detection of Herpes virus 1 and 2 genome

in amniotic fluid

Method:

Real Time PCR (quantitative amplification)

Principle:

To assess the presence of viral genome in amniotic fluid. Analysis

of the region 42


A39. Detection of Papilloma Virus genome

Method:

amplification of the microsatellite marker region-specific for

the MY9 and MY11 and fragment analysis on agarose gel

Principle:

to assess the presence of the viral genome

A40. Genotyping genome Papilloma Virus

Method:

amplification and fragment analysis with Reverse Dot Blot

Principle:

detection of the following genotypes: 16, 18, 31, 33, 35, 39, 45,

51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66, 69, 71, 6, 11 , 40, 43, 44,

54, 70

REMIND!!!

(#) ALL PRE-NATAL SAMPLES FOR MOLECULAR TEST ARE SUBJECTED TO M.C.C.

(Maternal Cell Contamination) EXCLUSION TEST BEFORE TO ANY TYPE OF FURTHER ANALYSIS

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