Polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) and PCR–restriction fragment length polymorphism (PCR-RFLP) are two independent methods used in the post-amplification genotyping of DNA variations. Both techniques are used in a wide range of screening applications to characterize single nucleotide polymorphisms (SNPs). The PCR-SSCP enables the identification of a potentially causative unknown SNP that could not be identified by PCR-RFLP. However, because complicated steps are not required to perform PCR-RFLP, it is used in many applications. On the other hand, PCR-RFLP is easier to process in terms of time and handover experience, the detection of a particular unknown SNP by PCR-SSCP has further chances. The simplicity of PCR-RFLP does not mean that it is better than PCR-SSCP. The reason is the limited ability of PCR-RFLP to detect nucleotide variations, which often go undetected because each restriction enzyme (RE) scans only a few recognition sequences, and other sequences are ignored. Furthermore, the efficacy of PCR-SSCP is sometimes hindered by many optimizations and also lack of experience. As PCR-SSCP allows other sequences within an amplicon to be separated and characterized, the choice between PCR-RFLP and PCR-SSCP is largely dependent on the reason for each genotyping experiment. This review provides a useful guide for comparing PCR-RFLP and PCR-SSCP in terms of their concepts, efficiency, ease of use, interpretation, and sensitivity as well as several other parameters. The comparison is extended to the practical applications of both techniques in terms of their utilization in molecular diagnostics and related applications.

Sanger dideoxy sequencing is vital in clinical analysis due to its accuracy, ability to analyze genetic markers like SNPs and STRs, capability to generate reliable DNA profiles, and its role in resolving complex clinical cases. The precision and robustness of Sanger sequencing contribute significantly to the scientific basis of clinical investigations. Though the reading of chromatograms seems to be a routine step, many errors conducted in PCR may lead to consequent limitations in the readings of AGCT peaks. These errors are possibly associated with improper DNA amplification and its subsequent interpretation of DNA sequencing files, such as noisy peaks, artifacts, and confusion between double-peak technical errors, heterozygosity, and double infection potentials. Thus, it is not feasible to read nucleic acid sequences without giving serious attention to these technical problems. To ensure the accuracy of DNA sequencing outcomes, it is also imperative to detect and rectify technical challenges that may lead to misinterpretation of the DNA sequence, resulting in errors and incongruities in subsequent analyses. This overview sheds light on prominent technical concerns that can emerge prior to and during the interpretation of DNA chromatograms in Sanger sequencing, along with offering strategies to address them effectively. The significance of identifying and tackling these technical limitations during the chromatogram analysis is underscored in this review. Recognizing these concerns can aid in enhancing the quality of downstream analyses for Sanger sequencing results, which holds notable improvement in accuracy, reliability, and ability to provide crucial genetic information in clinical analysis.

Sheep possess desirable characteristics for farmers such as resistance to disease, good acclimation to environmental fluctuations, and endurance to extreme weather. This may, in part, be due to hematology characteristics and various types of hemoglobin that interact differently to external changes; displaying various reproductive ability in certain environmental conditions. This review aims to investigate the effect of hematology parameters and hemoglobin types on the adaptation and reproductive performance of sheep. Hematological parameters are useful tools for the detection of adaptation and physiological traits of these animals. The reproduction of sheep has depended on the ability of each type and breed to acclimate to different environmental conditions. In addition, hemoglobin types have also been correlated with the environmental adaptability and physiological traits of sheep. Thus, the present review provides useful information on the association of hematology parameters and hemoglobin types in sheep with adaptation and reproductive performance, and could be used as vital tools that help in determining the species or breed of sheep that are more adapted to certain environments or have more reproductive potential.

This study was conducted to determine the genetic variation of the prolactin (PRL) gene in 5' flanking region and its role with several reproductive traits in Awassi sheep. One-hundred six Iraqi sexually mature and healthy Awassi ewes that aged between 2 and 2.5 years were included in this study. Ewes were classified into two the main divisions: ewes producing twins and ewes producing single offspring. Two genotypes (AA and AT) were observed in Awassi sheep. While genotype AA was detected in Awassi ewes that produced twins, genotype AT was detected in ewes that produced a single offspring. The sequencing reactions identified nine SNPs in the prolactin gene at the 5′ flanking region in Awassi sheep, differing from the prolactin reference sequence (GenBank accession number X16641.1). Genotype AT possessed one single nucleotide polymorphism SNP substitute comparison with the AA genotype in Awassi ewes. The association analysis revealed that the AA genotype is characterized by significantly higher levels of the progesterone concentration, twinning ratio, fecundity, and prolificacy than the AT genotype. In conclusion, a new SNP (g.1209 A>T) was discovered within the ovine flanking region which potentially influences prolactin gene expression. These results showed that the genotype AA associated with high prolificacy of Awassi sheep may be used as a selection criterion for improving the reproductive performance of Iraqi Awassi sheep.