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Human Genome Epidemiology: A Scientific Foundation for Using Genetic Information to Improve Health and Prevent Disease

Part II:
Methods and Approaches 1: Assessing Disease Associations and Interactions

Chapter 5 Tables

Methods for Assessing Genotypes in Human Genome Epidemiology Studies

Karen Steinberg and Margaret Gallagher

Table 5-1
Comparison of Specimens for DNA Banking for Epidemiologic Studies
Specimen Type DNA yield Advantages Disadvantages
Blood Spots

12 – 42 ng/μL (adults)a
43 – 78 ng/μL (neonates)a
1/4 inch punch from 75 μL volume yields about 12 μL of bloodb

 – small sample size
– ease of sample collection
– ease of shipping (regular mail)
– stability and low cost storage
– offers a source for study of exogenous or endogenous compounds other than DNA
– genotyping generally requires 10 ng/genotype, and with current technology as little as 2.5 ng per SNP so that scores to hundreds of genotypes could be obtained from one blood spot.		 – low DNA yield: may not be suitable for whole genome amplification.
– non-renewable
– smaller amplicons
Whole blood
(anticoagulated or blood clots)
Buffy coat		 100 – 400 μg/10 mLc
~200 μg/mLc		 – relatively low-cost storage (-80° C)
– yields large quantities of high-quality, genomic DNA
– offers a source for study of exogenous or endogenous compounds other than DNA		 – invasive sample collection
– shipping (special requirements)
– non-renewable
Transformed lymphocytes 106 cells = 6 μgc
1-2 x106 cells = 5-10 μg		 – renewable source of DNA
– yields large quantities of high-quality, genomic DNA		 – labor intensive preparation
– high-cost storage (liquid nitrogen-and periodic reculture)
– does not offer a source for study of exogenous or endogenous compounds other than DNA or RNA
Buccal cells 49.7 μg mean; 0.2 – 134 μg range (mouthwash- total DNA)d
12-60 μg range (mouthwash – total DNA)e
~16 – 30 μg median; 1 – 290 μg range (mouthwash- hDNA)f
32 μg median; 4 – 196 μg range (mouthwash- hDNA)g
1 – 1.6 μg/2 cytobrushes- median; 6 ng – 13 μg range (hDNA)e
1 – 2 μg/ cytobrush (total DNA)h 1 – 2 μg/ swab (total DNA)h		 – non-invasive collection
– ease of sample collection and shipping (allows participant to collect and mail specimen).
– genotyping generally requires 10 ng/genotype, and with current technology as little as 2.5 ng per SNP so that many thousands of genotypes could theoretically be obtained from a buccal cell specimen.		 – low DNA yield: not in general use for whole genome amplification;
– highly variable yield-does not offer a source for study of exogenous or endogenous compounds other than DNA or RNA
– bacterial contamination must be addressed

aRef. 5, bRef. 6, cRefs. 7-10, dRef. 11, eRef. 12, fRef. 13, gRef. 14, hRef. 1

 

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Table 5-2
Critical steps in molecular methods requiring quality control

Molecular method a Critical step a QC method
Nucleic acid amplification specimen acquistion

DNA isolation

primer performance

 identity check (e.g. microsatellites) and barcoding
PCR (Other methods such as ligase chain reaction and cloning are not included) DNA isolation

PCR reagents

Detection

 Assess yield by quantitation
Assess quality by electrophoresis, endonuclease digestion, PCR

Verify performance of reagents and primers

Monitor sensitivity and specificity using positive controls, standards, negative controls
a. Modified from CLIAC

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