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Developmental biology

 

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Julia Kerk

Semester: 11

 

Interest: Gynecology, Neonatology

 

Project description

 

Development of a multiplexed PCR system for simultaneous detection of four inflammation-associated single nucleotide polymorphisms

 

Intrauterine infection is associated with an increased risk of preterm delivery (1) and brain damage in preterm newborns (2). A fetal and/or maternal inflammatory response appears to play an important role in this etiologic scenario (3). Therefore, it seems likely that single-nucleotide-polymorphisms (SNPs) in inflammatory-associated genes are involved in both preterm birth (4, 5) and brain damage in preterm newborns (6).

Research that requires blood specimens for measurement of multiple biomarkers represent an ethical challenge in preterm infants due to the small amount of blood available. Therefore, miniaturized, multiplexed approaches are desirable for such studies.

In this study, we developed a multiplexed ARMS-based PCR assay (7) to identify four SNPs (TLR-4 (Asp299Gly); TNF-α (-308 G/A); IL-1beta (+3953 C/T) and IL-10 (-1081 G/A)) in a single specimen, applicable on dried blood spots.

 

Methods

 

DNA extraction, PCR, RFLP analysis, ARMS-PCR

 

Abbreviations

 

ARMS: amplification refractory mutation system

RFLP: restriction fragment length polymorphism

PCR: polymerase chain reaction

SNPs: single-nucleotide-polymorphisms

TNF-α: tumor necrosis factor-alpha

IL-1β: interleukin-1beta

IL-10: interleukin-10

TLR-4: toll like receptor-4

 

Literature

 

1. Goldenberg, R.L., J.C. Hauth, and W.W. Andrews, Intrauterine infection and preterm delivery. N Engl J Med, 2000. 342(20): p. 1500-7.

 

2. Bejar, R., et al., Antenatal origin of neurologic damage in newborn infants. I. Preterm infants. Am J Obstetr Gynecol, 1988. 159(2): p. 357-63.

 

3. Dammann, O. and A. Leviton, Inflammatory brain damage in preterm newborns - dry numbers, wet lab, and causal inference. Early Hum Dev, 2004. 79(1): p. 1-15.

 

4. Aidoo, M., et al., Tumor necrosis factor-alpha promoter variant 2 (TNF2) is associated with pre-term delivery, infant mortality, and malaria morbidity in western Kenya: Asembo Bay Cohort Project IX. Genet Epidemiol, 2001. 21(3): p. 201-11.

 

5. Genc, M.R., et al., Polymorphism in the interleukin-1 gene complex and spontaneous preterm delivery. Am J Obstet Gynecol, 2002. 187(1): p. 157-63.

 

6. Dammann, O., S.K. Durum, and A. Leviton, Modification of the infection-associated risks of preterm birth and white matter damage in the preterm newborn by polymorphisms in the tumor necrosis factor-locus? Pathogenesis, 1999. 1(3): p. 171-7.

 

7. Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucl Acids Res 1989; 17(7) :2503-16

 

Supervisors

 

Thilo Dörk

 

Olaf Dammann

V.i.S.d.P: Christiane Dammann, updated: 12.12.2006