Comprehensive GeneSequence Analysis fromBloodspot and Saliva DNA
T. Vo, J. Schymick, J. Marucut, A. Kammesheidt
Ambry Genetics l 100 Columbia #200 l Aliso Viejo, CA 92656 l www.ambrygen.com

Abstract
Ambry Genetics’ proprietary full gene sequence detection methods have been validated
for DNA isolated from blood spots and saliva, in addition to DNA from whole blood andcultured amniocytes used to date. Blood can be collected on Schleicher & Schuell (S&S)
903 specimen collection paper commonly used in newborn screening programs.
Alternatively, saliva can be collected into an OrageneTM DNA Self-Collection container(DNA Genotek Inc.). High quality DNA can be obtained from either starting material,with a half of a blood spot (0.5 inch diameter) or 250 ul saliva giving yields ofapproximately 1.5-4.5 ug and 8-12 ug DNA, respectively. The isolated DNA has beensuccessfully analyzed for sequence variations in the entire Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) coding sequence using the Ambry TestTM: CF, utilizing
modified temporal temperature gradient electrophoresis (mTTGE) and dye terminatorsequencing methods. In addition, DNA from bloodspots has been successfully analyzed
for sequence variations in the coding and flanking regions of the Beta Globin (HBB) geneusing a dye terminator sequencing method. We observed a PCR amplification templatesize limit of approximately 800 bp for DNA isolated from bloodspots. Analyticalsensitivities and specificities between the specimen types for these two genes were shown
to be similar. Furthermore, the specimens are intact for years in their collected media andthe isolated DNA are as stable as whole blood DNA when stored at –20oC or –80oC.

Figure 4: Sequence Analysis

Figure 1: Extracted Genomic DNA

A.The chromatographshows a sample
sequencing result fromwhole blood (W) versus
saliva (S) DNA templatefrom the same individual
at the beginning of exon
1 of HBB. Both templates
were 1914 bp long.

A. 4 ul of genomic DNA extracted from two halves of a blood spot (bs1+2) wereanalyzed on agarose gel along with 2 ul of DNA extracted from 1 ml of whole blood(B1-B6 and B8).
B.2 ul of genomic DNA extracted from 250 ul of saliva (S1 and S2) were analyzed on
agarose gel along with 2 ul of DNA extracted from 1 ml of whole bloods (B1 and B2).
To assess the DNA quantity and quality, 2 ul of 50 ng/ul and 100 ng/ul high molecularweight DNA (M) were also included in the gels. The saliva samples S1 and S2 represent
the maximum (~200 ng/ul) and minimum yields (~80 ng/ul) obtained from several saliva
samples. Most samples gave yields between ~80-120 ng/ul.

B.The same region inHBB exon 1 is shown insense and antisense
direction when synthesizedfrom bloodspot DNA,template A.

Figure 2: TTGE Comparison

By making blood spots an acceptable specimen type, Ambry Genetics can now offer
CFTR and HBB full gene sequence analysis to newborn screening programs, eliminating
the need for additional blood collection and allowing for more rapid processing ofsamples. In addition, clinics or individuals not having customary access to phlebotomists
can utilize the saliva kits to submit their DNA for comprehensive genetic analysis.

Blood SpotsC Whole Bloodbs1 bs2 bs3 bs4 bs5

B1B2

B3

B4C.The beginning ofHBB exon 3 is shown insense and antisense
direction when synthesizedfrom bloodspot DNA,
template B.

Introduction
Comprehensive gene sequence detection methods typically rely on ample amounts of DNA
isolated from whole blood for analysis. Our assays, in particular for cystic fibrosis (CF) andbeta globin (HBB) are often requested for newborns, babies and small children. Inaddition, specimens are sent from locations worldwide. We investigated if our methods
could be optimized for use with bloodspot DNA and saliva DNA in order to accommodate
a broader range of sample collection and shipping conditions. Advantages of providing
these options are time and cost savings, and expedited diagnoses.

A.TTGE gel showing 3 different PCR products amplified from CFTR gene using DNA
extracted from 5 blood spot (bs1-bs5) and 4 whole blood (B1-B4) samples. A common
polymorphism (cP) is detected in samples bs2 and bs3 and a polymorphic sequence (P) isdetected in samples bs4 and B4. Both of the sequence variants are included in the control(C) sample. Potential mutation (M) is observed in sample B1. The absent band could
result from a sequence variation where the PCR primers anneal or a homozygous deletionof the genomic sequence. Any sequence variations are verified by direct sequencing.
Whole BloodMethods

B1

B2

B3

B4CSaliva
S1

S2

S3

S4

S5

S6S7S8DNA extraction from blood spots: Blood was collected on FDA approved
Schleicher & Schuell (S&S) 903 specimen collection paper (1), and DNA was extracted
based on a method published by Walsh et al. (2). Briefly, half of a dried blood spot wasincubated and rinsed three times in deionized water prior to cell centrifugation. A small
volume of freshly made 5% Chelex 100 resin was then added. The suspension wasincubated at 56oC for 2 hours, then boiled for 8 minutes, and centrifuged at high speed.
The DNA supernatant was removed from the resin pellet and used for PCR.
DNA extraction from saliva: Two milliliters (4-5 spits) of saliva were collected into a
DNA Genotek’s OrageneTM DNA Self-Collection container, which contains ~2 ml of cell
lysis solution, and kept at room temperature. Saliva samples were processed according tomanufacturer specifications (3). Briefly, Oragene Purifier solution (proprietary proteinaseand precipitation solution) was added to the saliva mixture, and incubated at 50oC for atleast 1 hour prior to precipitation with ethanol. The DNA pellet was air dried anddissolved in 1X TE. The DNA was then used for PCR.
DNA quality and quantity assessments: The extracted DNA was analyzed on
agarose gels with known amount of high molecular weight DNA to assess DNA qualities
and quantities. DNA was tested with PCR amplification procedures and primers used for
CFTR TTGE and sequencing analysis (4). TTGE: For bloodspots, 10-30 ng input DNA(2 ul) was used with HotStarTaq Master Mix (Qiagen) per 10 ul reaction. Typical PCRconditions were 1 cycle: 95oC/ 15 min, 39 cycles: 94oC/ 30 sec, 49-62oC/ 30 sec,
72oC/ 30 sec, 1 cycle: 72oC/ 10 min. PCR products were then processed for TTGE on
DCode gels (BioRad) in adherence with the Ambry Test technology. For saliva DNA,~40-60 ng input DNA (0.5 ul) was used per 10 ul reaction, identical to the standard
method for whole blood, and PCR amplification was performed for 35 cycles.
Sequencing: For bloodspots, 10-30 ng input DNA and for saliva, 40-60 ng DNA wereused per 10 ul PCR reactions to amplify template for subsequent dye terminator
sequencing. Exons were amplified with unique primer sets using Taq PCR Master Mix(Qiagen, Valencia CA). Typical PCR conditions were 1 cycle: 95oC/ 5 min, 35 cycles:
94oC/ 30 sec, 50-55oC/ 30 sec, 72oC/ 30-45 sec, 1 cycle: 72oC/ 10min. For HBB
sequencing, the template length was kept under 800 bp. Dye terminator sequencing wasset up using Beckman DTCS reagents and templates were analyzed on a CEQ8000.

B.TTGE gel showing 4 different PCR products amplified from CFTR gene using DNA
extracted from 4 whole blood (B1-B4) and 8 saliva (S1-S8) samples. A common
polymorphism (cP) is detected in saliva S7 and control (C) samples. The control sample
also contains a mutation (M) located in the bottom PCR fragment.

Figure 3: Sequence Templates

A.The agarose gel shows amplification product for three different HBB templates
synthesized for sequencing. The DNA was amplified with PCR to yield a 1914 bp
fragment for the Beta-Hemoglobin gene (HBB) containing exons 1, 2 and 3. Larger
templates were more difficult to generate from bloodspot DNA. Thus, the HBB sequencing
templates were generated in 2 separate reactions giving rise to two smaller PCR
templates of 771 bp (A, covering exons 1 and 2), and a 682 bp template (B, coveringexon 3). We have observed a consistent drop in yields for templates over 800 bp forbloodspot DNA and design template lengths accordingly. (Bs) refers to DNA extracted
from a blood spot sample; (S) to DNA extracted from a saliva sample; (Ca) to DNA
extracted from a cultured amniocyte sample; and (Wb) to DNA extracted from a whole
blood sample.

Results• DNA extraction methods for half of a blood spot (0.5 inch diameter) and ~250 ul ofsaliva typically gave yields of ~300 ul of 5-15 ng/ul (1.5-4.5 ug total) and ~100 ul of
80-120 ng/ul (8-12 ug total) of DNA, respectively.
• Bloodspot DNA was of high quality, although PCR products generated from blood spots
generally need to be less than 800 bp to amplify reliably.
• In order to obtain sufficient PCR product from bloodspot DNA for TTGE analysis, PCR
amplification cycles were increased by 4 cycles. Sequencing analysis did notnecessitate amplification cycle changes compared to whole blood DNA. The assays’
analytical sensitivities and specificities were unchanged.
• DNA yield from ~250 ul of saliva is similar to yield from 1 ml of whole blood, andsimilar performance on TTGE and sequencing protocols were observed.

Conclusion• Ambry Genetics’ proprietary full gene sequence detection method utilizing a
combination of TTGE scanning and sequencing has been validated for DNA extracted
from blood spots and saliva specimens.
• Analytical sensitivities and specificities for blood spot and saliva DNA are similar to
those of whole blood DNA in The Ambry Test: CF and in The Ambry Test: HBB.
• Blood spots collected as part of newborn screening programs now can be used inAmbry Genetics’ CFTR and HBB full gene sequence analysis when follow-up molecularstudies are needed, thus avoiding multiple blood collection and office visits, while
expediting diagnosis. Potentially, DNA from stored bloodspots of deceased patients
could be evaluated to help establish diagnoses and complete pedigrees.
• Clinics or individuals without customary access to phlebotomists can utilize theOragene saliva kits to submit their DNA to Ambry Genetics for CFTR and HBB full genesequence analysis.

References
1. www.schleicher-schuell.com
2. Walsh, PS, Metzger, DA, and Higuchi R. Chelex 100 as a medium for simpleextraction of DNA for PCR-based typing from forensic material. Biotechniques 10(4):506-513, 1991.
3. DNA Genotek’s OrageneTM DNA Purification protocol, DNA Genotek Inc., Ottawa,Ontario, Canada. www.dnagenotek.com, (613) 723-5757.
4. Danziger KL, Black LD, Keiles SB, Kammesheidt A, Turek PJ.– Improved detection ofcystic fibrosis mutations in infertility patients with DNA sequence analysis. HumReprod. 2004 Mar;19(3):540-6. Epub 2004 Jan 29.