Gene Link

Technical Sheet
q Catalog No. 40-2002-10 Rh D 10 nmoles
q Catalog No. 40-2003-10 Rh CcEe 10 nmoles

Shipped at ambient temperature. Store at -20^oC
For research use only.
Not for use in diagnostic procedures; for clinical purposes only.

Background

The Rh based blood grouping is termed positive or negative based on the presence or absence of the D antigen. Rh alloimmunization in Rh negative pregnant women is of concern because of the potential for the fetus to develop hemolytic disease of newborns and autoimmune diseases. Anemia leading to hydrops, perinatal death, or both occurs in 25% of fetuses sensitized to the RhD antigen in the absence of optimal management. In utero diagnosis and treatment considerably improves the condition with survival rates greater than 75% in severely affected fetuses. However these invasive therapies may be unnecessary in some cases if the fetal Rh status were known prenatally.

A method of determining fetal Rh status early in pregnancy is now possible by DNA analysis of amniotic cells (1). The Rh blood group locus consists of two related structural genes, D and CcEe. These highly homologous genes which share greater than 96% identity in their coding region have been cloned and the molecular basis of the Rh blood group established (2). The RhD-positive and RhD-negative polymorphism is associated with the presence or the absence of the D gene (there is no ‘d’ gene). The C/c and E/e antigens are encoded by a unique gene. The E/e associated nucleotide polymorphism results in one amino acid substitution at position 226 (proline to alanine), whereas the C/c antigenic polymorphism consists of six nucleotide substitutions leading to four amino acid changes at position 16 (Cys16Trp), 60 (Ile60Leu), 68 (Ser68Asn) and 103 (Ser103Pro).

DNA analysis for Rh genotype loci specifically amplifies DNA fragments for the RhD and RhCcEe gene. Due to the high sensitivity and specificity of the test at the DNA level, occasionally the results may not match those obtained by serologic methods. The test will type individuals as RhD positive who are D^u low grade status serologically (3-5), this is due to the absence of the gene product at the protein level due to partial deletions. The total error rate should be less than 1%.

Material Supplied

Two lyophilized oligonucleotide primers are supplied. Please refer to item number on the top of this sheet (Rh D or Rh EeCe Genemer>ä ). Each tube contains 10 nmoles of the primer. The quantity supplied is sufficient for 400 regular 50>m l PCR reaction.

Reconstitution

Stock solution: Add 50>m l sterile water to each tube containing the primer. The 10 nmoles of primer when dissolved in 50>m l water will give a solution of 200 >m Molar i.e. 200 pmoles/>m l.
Primer Mix: Prepare a 10 pmoles/>m l Primer Mix solution containing BOTH the primers. Example; add 180 >m l water to a new tube. To this tube add 10 >m l of EACH of the primer stock solution from #1 step above. Label this tube as Primer Mix 10 pmoles/>m l.

Protocol for RhD and Rh EeCc Genotyping
The following PCR* profile has been optimized for Rh D and Rh EeCc specific product amplification using the supplied Genemers>ä

PCR reaction (100 >m l)

Denaturation 94oC 30 sec.

Annealing 49oC 1 min.

Elongation 72oC 1 min.

30 cycles; 7 min. at 72oC, soak at 4oC.

Precipitate products, dissolve in 10 >m l water, add 2 >m l loading buffer.

Electrophoresis
Load samples in a 1.8 % agarose gel or a 3% Nusieve agarose gel. Run at 90 mAmps for 2.5 hrs.

Results and Interpretation
The primers A1/A2 will give 136 bp PCR product specific for Rh EeCc gene exon 7 (Catalog No. 40-2003-10) and primers A3/A4 will give 186 bp PCR product specific for Rh D gene exon 10 (Catalog No. 40-2002-10). Can perform multiplex of A1/A2 & A3/A4 as shown in the figure below

RhD negative- 136 bp only;
RhD positive- both 136 & 186 bp.

  1  2  3   4  B   5  6  7

Figure. Lane 1 is molecular weight markers.

Lanes 2-4 represent PCR from a Rh D positive DNA and Lanes 5-7 represent PCR from Rh D negative DNA.

Lanes 2, 5 & 7 PCR product of A1/A2 amplification, lanes 3 from A3/A4 amplification.

Lane 4 is a multiplex of A1/A2 and A3/A4 amplification.

References
1. Bennet, P.R., et al. (1993) Prenatal determination of fetal RhD type by DNA amplification. NEJM 329:607-610.
2. Mouro, I., et al. (1993) Molecular genetic basis of the human Rhesus blood group system. Nature Genetics 5:62-65.
3. Simsek, S., Bleeker, P.M., Borne, A.. E.G. (1994) Prenatal determination of fetal RhD type. NEJM 330:795.
4. Bennet, P., Warwick,R. and Carton, J-P. (1994) Prenatal determination of fetal RhD type. NEJM 330:795-796.
Westhoff,C.M. and Wylie, D.E. (1994) Identification of a new RhD-specific mRNA from K562 cells. Blood 84:3098-3100

**The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.
All Gene Link products are for research use only.

Appendix

PCR Premix preparation
Typical Premix
-----------------------------------------/50 >m l----rxn /1ml
10 x PCR Buffer ----------------___4.5 >m l___ 100>m l
dNTP mix (2.5mM each) _________4 >m l___ 100>m l
Primer Mix (10 pmol/>m l each) ___2.5 >m l____ 63>m l
(25 pmol of each primer/50>m l)
Sterile water _________________-34 >m l___ 737>m l
___________________________----------__--------
Total ________________________45 >m l____ 1ml

Nucleotide Dilution
Stock:____100 mM; Prepare a final diluted 2.5 mM solution
Preparation
Each 100 mM dNTP _______125 >m l (Total 500 >m l)
Water ____________________________4.5 ml
_________________________________----------

Total volume ________________________5.0ml

Taq Premix (per 50>m l reaction, scale up as required)

10 x PCR Buffer ___________0.5>m l
Taq polymerase ___________-0.25>m l
Sterile water______________- 4.25>m l
________________________-----------
_________________________5>m l/rxn.

PCR reaction (50>m l)
Diluted DNA(100ng/>m l) ______-1 >m l
PCR premix _______________45 >m l
Taq premix _________________5 >m l

PCR products post-processing
1. For oil layered PCR only. Add 200>m l of CHCl3 to each tube, vortex and spin.
2. Transfer the upper aqueous layer to a fresh eppendorf tube, add 1/10 volume of 3M NaAc (pH 5.2), and 2 volumes of absolute ethanol, precipitate DNA at -80oC for 10 minutes.
3. Spin, rinse the DNA pellet with 700>m l of 75% ethanol and dry the pellet in the speedvac.
4. Dissolve the pellet in adequate amount of TE.

Ordering Information

GENEMER>ä

Product

Size

Catalog No.

Price, $

Sickle Cell SC2/SC5 primer pair

10nmoles

40-2001-10

100.00

RhD (Rh D gene exon 10 specific)

10nmoles

40-2002-10

100.00

Rh EeCc (Rh Ee and Cc exon 7 specific)

10nmoles

40-2003-10

100.00

Fragile X (spanning triple repeat region)

10nmoles

40-2004-10

100.00

Gaucher 1226G mutation specific

10nmoles

40-2005-10

100.00

Gaucher 1448C mutation specific

10nmoles

40-2006-10

100.00

Gaucher 84GG mutation specific

10nmoles

40-2007-10

100.00

Gaucher IVS2 mutation specific

10nmoles

40-2008-10

100.00

Cystic Fibrosis >D F508

10nmoles

40-2009-10

100.00

Cystic Fibrosis G542X

10nmoles

40-2010-10

100.00

Cystic Fibrosis W1282X

10nmoles

40-2011-10

100.00

Cystic Fibrosis G551D/R553X

10nmoles

40-2012-10

100.00

Cystic Fibrosis N1303K

10nmoles

40-2013-10

100.00

Cystic FibrosisCT3849

10nmoles

40-2014-10

100.00

SRY (sex determining region on Y)

10nmoles

40-2020-10

100.00

X alphoid repeat

10nmoles

40-2021-10

100.00

Y alphoid repeat

10nmoles

40-2022-10

100.00

Please inquire about other GENEMER>ä not listed here

LINKMER>ä

LINKMER>ä are primer pairs for specific amplification of chromosomal sequences; specially suited for linkage analysis and chromosomal mapping. LINKMER>ä are supplied as lyophilized powder in aliquots of 10nmoles. The 10nmoles of primer when dissolved in 500>m l sterile water or TE will give a solution of 20>m Molar i.e. 20pmoles/>m l. The quantity supplied is sufficient for at least 400 regular 25>m l PCR reaction.

Product	Size	Catalog No.	Price
Please inquire, Linkmers>ä	10nmoles		$75.00

**The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.

GENEMER>ä
Product	Size	Catalog No.	Price, $
Sickle Cell SC2/SC5 primer pair	10nmoles	40-2001-10	100.00
RhD (Rh D gene exon 10 specific)	10nmoles	40-2002-10	100.00
Rh EeCc (Rh Ee and Cc exon 7 specific)	10nmoles	40-2003-10	100.00
Fragile X (spanning triple repeat region)	10nmoles	40-2004-10	100.00
Gaucher 1226G mutation specific	10nmoles	40-2005-10	100.00
Gaucher 1448C mutation specific	10nmoles	40-2006-10	100.00
Gaucher 84GG mutation specific	10nmoles	40-2007-10	100.00
Gaucher IVS2 mutation specific	10nmoles	40-2008-10	100.00
Cystic Fibrosis >D F508	10nmoles	40-2009-10	100.00
Cystic Fibrosis G542X	10nmoles	40-2010-10	100.00
Cystic Fibrosis W1282X	10nmoles	40-2011-10	100.00
Cystic Fibrosis G551D/R553X	10nmoles	40-2012-10	100.00
Cystic Fibrosis N1303K	10nmoles	40-2013-10	100.00
Cystic FibrosisCT3849	10nmoles	40-2014-10	100.00
SRY (sex determining region on Y)	10nmoles	40-2020-10	100.00
X alphoid repeat	10nmoles	40-2021-10	100.00
Y alphoid repeat	10nmoles	40-2022-10	100.00

Please inquire about other GENEMER>ä not listed here