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HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Refer to Figure 1 for a schematic representation of the procedures involved in DNA-RFLP analysis. Two alternative methods are detailed below; the choice of method depends upon the probe-labeling and detection system preferred. For 32P-labeled probes and autoradiographic detection, refer to the first five sections. For digoxigenin-AMPPD chemiluminescent detection, refer to the first three and the sixth section.
Immunology of Insulin-Dependent Diabetes Mellitus
Published in Irun R. Cohen, Perspectives on Autoimmunity, 2020
Christian Boitard, H. O. McDevitt
Characterization of the class II allelic gene products associated with IDDM have relied in the past on serological techniques. The associations defined are partial, and the majority of subjects with susceptibility alleles never develop IDDM. In addition, 10% of IDDM patients do not carry the susceptibility alleles defined by serological markers. Diseases which do not appear to be related to IDDM are associated with identical susceptibility alleles. Serological definition of susceptibility alleles, therefore, may be incomplete and/or insufficient. Restriction fragment length polymorphism (RFLP) is a first step in characterizing, at the DNA level, susceptibility alleles and in verifying whether alleles which have different amino acid sequences but share identical antigenic determinants are detected as different by RFLP. This approach has been developed using a DR β probe to study HLA-DR in DR4 and DR3 heterozygous individuals. An increased frequency of a Pstl 18-kilobase (kb) fragment has been observed.176 Similar studies in DR2 healthy and diabetic individuals using a DC β cDNA probe have also allowed the definition of a 2.2-kb fragment which may be related to protection against IDDM.177
A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
A 1000-fold increase in the sensitivity of the method is achieved using a modified primer in the PCR, thereby introducing anartificial restriction siteinto the amplified product. This creates an RFLP, indicative of a given mutation, that can be used for screening purposes or for monitoring the effects of therapy in oncology.
Evaluation of genetic polymorphisms at 21 autosomal STR loci in Ramgharia Sikh population of Punjab, India
Published in Annals of Human Biology, 2022
Amandeep Kaur Bhambara, Abhishek Singh, Vivek Sahajpal, Mukesh Thakur, Deepika Bhandari, Shivkant Sharma, Mukesh Kumar Thakar
India, being a diverse country with an accounted population of about 1.2 billion (Census of India, 2011), portrays a significantly colourful canvas comprising of novel assimilation of various ethnic groups differing in terms of their communities, cultures, and religions. Several researchers and philosophers have been engrossed for centuries in studying the diverse patterns regarding human appearances and forms. The genetic variation among human populations was first reported in 1919 by Ludwik and Hanka Hirszfeld, pioneers in the field of blood typing (Allan 1963). However, the variations studied in blood groups were not sufficient to identify a particular individual from a gene pool. Thus, this area of interest was revolutionised by detecting variations in the human gene pool at the DNA level (Kandpal et al. 2011). In the early days, RFLP (Restriction Fragment Length Polymorphism) analysis was employed for DNA analysis followed by the PCR-based assays e.g. SNPs, VNTRs, and Short Tandem Repeats (STRs). By the mid-1990s, forensic DNA testing introduced multiple STR markers in single multiplexed reactions (Gusmão et al. 2006).
The relationship between KRAS LCS6 polymorphism and endometrium cancer
Published in Journal of Obstetrics and Gynaecology, 2020
Feyza Nur İncesu Çintesun, Özlem Seçilmiş Kerimoğlu, Ersin Çintesun, Süleyman Nergiz, Hasan Acar, Çetin Çelik
The Restriction Fragment Length Polymorphism (RFLP) method is a method which allows the examination of DNA profiles formed by separation in the agarose gel electrophoresis system of DNA segments and fragmentation with a restriction endonuclease enzyme specific to genomic DNA. By identifying specific sequences in the DNA of these enzymes, which are found in bacteria, the fragmentation process occurs in the identified region or in another specific sequence outside this region. Genotyping of single nucleotide polymorphisms occurs in two steps with this method. The first step is based on PCR product sequencing or enzyme fragmentation after gel electrophoresis for the genotyping of the polymorphic region by hundreds of thousands of proliferations with PCR of approximately 200 base pairs around the single nucleotide polymorphism (SNP) in the DNA region (Linn and Arber 1968).
VAL158MET catechol O-methyltransferase polymorphism contributes to the development of preeclampsia
Published in Hypertension in Pregnancy, 2020
Tamara Sljivancanin Jakovljevic, Olivera Kontic-Vucinic, Nadja Nikolic, Jelena Carkic, Jelena Milasin
DNA was extracted from peripheral leukocytes using the salting‐out method (23). Analysis of COMT SNP at position 158 in the coding sequence were performed using Polymerase Chain Reaction (PCR) and Restriction fragments length polymorphism (RFLP) analysis. PCR reaction was performed in a mixture volume of 25 μL containing 13 μL of 2x PCR Master Mix, 200 nM of each primer, water, and 3 μL of DNA sample. Specific primers used for the amplification of the sequence surrounding COMT Val158Met SNP were: forward primer – 5ʹ-ACTGTGGCTACTCAGCTGTG-3ʹ and reverse primer – 5ʹ-CCTTTTTCCAGGTCTGACAA-3ʹ. PCR temperature profile was: initial denaturation at 95℃ for 5 min, 35 cycles – at 95℃ for 30 sec, then 55℃ for 30 sec and 72℃ for 30 sec, and final extension at 72℃ for 7 min. A 169 bp long PCR product was obtained. RFLP analysis was conducted using Nla III (Thermo Scientific), a fast digest restriction enzyme. In the absence of the variant allele, the enzyme generated DNA fragments of 114, 29, and 26 bp, whereas in the presence of the SNP PCR product digestion gave fragments of 96, 29, 26, and 18 bp.