title: Detection of α-globin gene deletions using denaturing high-performance liquid chromatography and multiplex ligation-dependent probe amplification.
abstract: BACKGROUND
  Multiplex ligation-dependent probe amplification (MLPA) has been used to detect deletions and mutations of the α-globin gene for diagnosis of α-thalassemia. MLPA reaction products are usually separated and analyzed by high-voltage capillary gel electrophoresis (CGE). The goal of this study was to find and use a cost-effective method to separate and analyze MLPA products.
  
  
  METHODS
  Blood samples were collected from China. DNA was extracted and amplified by PCR using fluorescently labeled primers. In this study, denaturing high-performance liquid chromatography (DHPLC) was used to separate and analyze the reaction products. And the optimal separation conditions were determined using nondenaturing columntemperature.
  
  
  RESULTS
  The DHPLC conditions were optimized and have been applied to separate MLPA products and 27 of the MLPA products from 50 to 320 bp were well separated. DHPLC was able to separate up to 37 reaction products that differed by 4-12 base pairs and detected target gene deletions by differences in peak size. Compared with CGE, both the specificity and sensitivity of DHPLC for the 107 DNA samples were 100%.
  
  
  CONCLUSIONS
  DHPLC could be used to test routinely for α-globin gene mutations and deletions. Combined with MLPA, DHPLC is a low-cost, simple to use, accurate technique with practical value.
authors.affiliation: Laboratory Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People's Republic of China.
authors.forename: Meiting
authors.initials: M
authors.lastname: Cao
authors.forename: Zhaolan
authors.initials: Z
authors.lastname: Liu
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Jia
authors.forename: Ning
authors.initials: N
authors.lastname: Tang
authors.forename: Ren
authors.initials: R
authors.lastname: Cai
authors.forename: Lirong
authors.initials: L
authors.lastname: Wang
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Bai
authors.initials: B
authors.lastname: Xiao
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Jingzhong
authors.initials: J
authors.lastname: Liu
mesh_terms: D000510:Alpha-Globulins / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D017353:Gene Deletion; D006801:Humans; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques; D017085:alpha-Thalassemia / Q000175:diagnosis*
chemical_list: D000510:Alpha-Globulins; D017931:DNA Primers; D004247:DNA
references.citation: Exp Mol Pathol. 2011 Aug;91(1):429-33
references.pmid: 21565183
references.citation: Nucleic Acids Res. 2002 Jun 15;30(12):e57
references.pmid: 12060695
references.citation: Hemoglobin. 2004;28(4):325-33
references.pmid: 15658188
references.citation: J Med Genet. 2005 Dec;42(12):922-31
references.pmid: 15894596
references.citation: Prenat Diagn. 2007 May;27(5):404-8
references.pmid: 17286309
references.citation: Hum Mutat. 2001 Jun;17(6):439-74
references.pmid: 11385705
references.citation: Ann Hematol. 2011 Jan;90(1):125-6
references.pmid: 20461518
references.citation: Hemoglobin. 2008;32(6):561-71
references.pmid: 19065334
references.citation: Transl Res. 2010 Mar;155(3):148-55
references.pmid: 20171600
references.citation: Blood. 1989 Apr;73(5):1081-104
references.pmid: 2649166
references.citation: Eur J Hum Genet. 2005 Feb;13(2):171-5
references.pmid: 15483643
references.citation: Clin Biochem. 2008 Jun;41(9):681-7
references.pmid: 18339318