Chain Reaction-mediated Site-directed Mutagenesis Research Articles

Alpha-1-antitrypsin deficiency in Madeira (Portugal): The highest prevalence in the world

Alpha-1-antitrypsin (AAT) deficiency is a common genetic disease which affects both lung and liver. Early diagnosis can help asymptomatic patients to adjust their lifestyle choices in order to reduce the risk of Chronic Obstructive Pulmonary Disease (COPD). The determination of this genetic deficiency prevalence in Madeira Island (Portugal) population is important to clarify susceptibility and define the relevance of performing genetic tests for AAT on individuals at risk for COPD. Two hundred samples of unrelated individuals from Madeira Island were genotyped for the two most common AAT deficiency alleles, PI*S and PI*Z, using Polymerase Chain Reaction-Mediated Site-Directed Mutagenesis. Our results show one of the highest frequencies for both mutations when compared to any already studied population in the world. In fact, PI*S mutation has the highest prevalence (18%), and PI*Z mutation (2.5%) was the third highest worldwide. The frequency of AAT deficiency genotypes in Madeira (PI*ZZ, PI*SS, and PI*SZ) is estimated to be the highest in the world: 41 per 1000. This high prevalence of AAT deficiency on Madeira Island reveals an increased genetic susceptibility to COPD and suggests a routine genetic testing for individuals at risk.

Rare codon clusters at 5′-end influence heterologous expression of archaeal gene in Escherichia coli

Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_ gnaD and Tac_ gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_ gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus ® RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_ gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_ gnaD and Tac_ gnaD gene sequences revealed that the 5′-end of the Sso_ gnaD sequence was rich in AGA arg and ATA Ile codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_ gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.

An Unexpected Product From Polymerase Chain Reaction-Mediated Site-Directed Mutagenesis Due to Misalignment of the Mismatched Primer.

Background: The I1307K (T3920 --> A) variant of the APC gene has been identified as a potential risk factor for colorectal cancer and is present in 6% of Ashkenazi Jews. Screening for this mutation may allow identification of people at elevated risk who would benefit from increased surveillance. Methods and Results: We designed an assay to detect the T3920 --> A allele using a primer mismatched at the 3 < 9 terminal nucleotide in the polymerase chain reaction (PCR) to generate a recognition site for the restriction enzyme Mse I. After optimization of the PCR for magnesium ion concentration and annealing temperature, the amplicon did not cut completely with the restriction enzyme in each of four tested DNAs. Sequence analysis of the PCR product that was resistant to digestion revealed that the T3920 --> A variant was not present. The artifact was caused by a single nucleotide loop-out in the genomic DNA template under the 3 < 9 region of the primer, which allowed the 3 < 9 terminal base of the primer to hybridize properly. As a result, the mismatched primer created a modified product different from that originally planned. At a magnesium ion concentration below the optimum for product yield, most of the product was digested by Mse I. Sequence analysis showed that, under these conditions, the intended product was produced. Conclusions: Mismatched primers can produce unintended products in a PCR due to looping out of a nucleotide in the template or the primer. The magnesium ion concentration can influence the sequence and amount of the product.

Novel germline mutations in the BRCA1 and BRCA2 genes in Indian breast and breast‐ovarian cancer families

The two major hereditary breast/ovarian cancer predisposition tumor suppressor genes, BRCA1 and BRCA2 that perform apparently generic cellular functions nonetheless cause tissue-specific syndromes in the human population when they are altered, or mutated in the germline. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast and/or ovarian cancers in the Indian population. We have screened for mutations the entire BRCA1 and BRCA2 coding sequences, and intron-exon boundaries, as well as their flanking intronic regions in sixteen breast or breast and ovarian cancer families of Indian origin. We have also analyzed 20 female patients with sporadic breast cancer regardless of age and family history, and 69 unrelated normal individuals as control. Thus a total of 154 samples were screened for BRCA1 and BRCA2 mutations using a combination of polymerase chain reaction-mediated site directed mutagenesis (PSM), polymerase chain reaction-single stranded conformation polymorphism assay (PCR-SSCP) and direct DNA sequencing of PCR products (DS). Twenty-one sequence variants including fifteen point mutations were identified. Five deleterious pathogenic, protein truncating frameshift and non-sense mutations were detected in exon 2 (c.187_188delAG); and exon 11 (c.3672G>T) [p.Glu1185X] of BRCA1 and in exon 11 (c.5227dupT, c.5242dupT, c.6180dupA) of BRCA2 (putative mutations - four novel) as well as fourteen amino acid substitutions were identified. Twelve BRCA1 and BRCA2 missense variants were identified as unique and novel. In the cohort of 20 sporadic female patients no mutations were found.

BRCA1 germline mutations in Indian familial breast cancer.

Germline mutation analysis of BRCA1 gene has demonstrated significant allelic heterogeneity. These differences represent historical influences of migration, population structure and geographic or cultural isolation. To date, there have been no reports of Indian families with mutations in BRCA1. We have screened for mutations in selected coding exons of BRCA1 and their flanking intron regions in three breast or breast and ovarian cancer families with family history of three or more cases of breast cancer under age 45 and/or ovarian cancer at any age. We have also analyzed 10 female patients with sporadic breast cancer regardless of age and family history, as well as 50 unrelated normal individuals as controls. Thus a total of 90 samples were analyzed for BRCA1 mutations using polymerase chain reaction-mediated site directed mutagenesis (PSM) and single stranded conformation polymorphism (SSCP) analysis for various selected exons followed by sequencing of variant bands. Eight point mutations were identified. Two deleterious pathogenic, protein truncating non-sense mutations were detected in exon 11 (E1250X) and exon 20 (E1754X) and six novel and unique amino acid substitutions (F1734S, D1739Y, V1741G, Q1747H, P1749A, R1753K). One complex missense mutation of exon 20 [V1741G; P1749A] was seen in two out of three families and another complex combination of missense and non-sense mutations of the same exon [V1741G; E1754X] was observed in only one family. These complex mutations exist only in breast cancer families but not in control populations of women. Three splice site variants (IVS20+3A>C, IVS20+4A>T, IVS20+5A>T) and two intronic variants (IVS20+21_22insG, IVS20+21T>G) were also detected. In the group of 10 sporadic female patients no mutations were found.

An unexpected product from polymerase chain reaction-mediated site-directed mutagenesis due to misalignment of the mismatched primer

An unexpected product from polymerase chain reaction-mediated site-directed mutagenesis due to misalignment of the mismatched primer

Polymerase chain reaction-mediated site-directed mutagenesis detection of Z and S alpha-1-antitrypsin alleles in family members

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder with a reduction in serum A1AT levels. In a large family, we used a polymerase chain reaction (PCR)-mediated, site-directed mutagenesis assay to detect the two most common A1AT deficient variants, Z and S. By coamplification, using primers for both the Z and S mutations, we were able to detect heterozygous and homozygous genotypes for both mutations in a single reaction. We compared our results with phenotype studies obtained by standard immunofixation and isoelectric focusing techniques at two reference laboratories. Whereas PCR and isoelectric focusing agreed completely, there were five discrepancies in the results obtained by the immunofixation procedure. The reference laboratory that provided these discrepant results later informed us of a quality control problem that accounted for their error. The family study included 12 individuals representing three generations. Two individuals were MM homozygotes, three were MZ heterozygotes, four were MS heterozygotes, and three were SZ heterozygotes. A thirteenth family member was diagnosed as a ZZ homozygote at another institution. We have shown that this PCR coamplification technique provides accurate information about the M, S, and Z alleles that is at least as useful as current reference laboratory methodologies.

A new test for detection ot the His 58 variant transthyretin allele in hereditary amyloidosis; creation of diagnostic restriction endonuclease recognition sites by PCR based mutagenesis

A new test has been developed to detect the His 58 variant transthyretin allele associated with familial amyloidotic polyneuropathy type II in the Maryland/German kindreds. Since the point mutation identified in this disease does not alter the restriction pattern of the TTR gene, previous DNA testing had been accomplished through the use ofallele-specific amplification. In order to increase the accuracy of the testing process and allow detection of individuals homozygous for the mutation, an alternative technique utilizing polymerase chain reaction-mediated site-directed mutagenesis has been developed to produce a unique restriction site in the PCR product derived from the His 58 variant allele. This method, which we have termed PCR-In-duced Mutation Restriction Analysis (PCR-IMRA), maybe applied to the detection of nearly all single base changes in genomic DNA which fail to produce a restriction fragment length polymorphism. A strategy for devising diagnostic tests by this method is presented.

Detection of a point mutation in cholesteryl ester transfer protein gene by polymerase chain reaction-mediated site-directed mutagenesis

We describe a method for the rapid and non-radioactive examination of DNA samples for a mutation of cholesteryl ester transfer protein using a polymerase chain reaction-mediated site-directed mutagenesis. CETP deficiencies were studied in 554 Japanese subjects (370 men, 184 women) aged between 18 and 91 (mean 48.3 years). By this method, we detected one homozygote and 3 heterozygotes of the CETP deficiency.

Detection of α1-Antitrypsin Z and S Mutations by Polymerase Chain Reaction-Mediated Site-Directed Mutagenesis

alpha 1-Antitrypsin (A1AT) deficiency is a relatively common autosomal recessive disease, resulting most often from a single base pair (1 bp) substitution called the Z mutation. Previous genetic tests for carriers and affected patients have relied on quantitative binding of radioactive probes to an amplified gene product, because the mutation does not occur within a restriction enzyme site. Using polymerase chain reaction (PCR)-mediated site-directed mutagenesis, one can introduce a base substitution near the mutation site, such that an inexpensive restriction enzyme (Taq I) can be used to differentiate normal subjects, carriers, and affected patients. We have applied this method to the detection of the Z mutation and the S mutation, which in heterozygotes with a Z allele may lead to the development of symptoms similar to those in ZZ homozygous subjects.

Detecting multiple cystic fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis

The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.