DNA Cryptography using DNA Fragment Assembly and Fragment Key Expansion for Genomic Data

This research proposes a tweaked scheme based on DNA fragment assembly to improve protection over insecure channel. The proposed procedure utilizes binary coding to change over an underlying plaintext into a reference DNA arrangement to deal with the fragmentation. DNA fragment key expansion is applied over the reference DNA sequence to make the short-chain fragments. The redundancy in the long-chain of reference DNA is removed using DNA fragment assembly. A look-up table is generated to store the binary values of overlapped fragments to be reassembled during the encryption and decryption processes to prevent artefacts. Also, it is used in an overlapped sequence to counteract cipher decomposition. The results and comparisons demonstrate that the proposed scheme can balance the three most important characteristics of any DNA masking scheme: payload, capacity, and BPN. Moreover, the potential for cracking the proposed tweaked method is more complex than the current strategies.

An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby

An albino infant wallaby was born to a mother with the wild-type body color. PCR and sequencing analyses of <i>TYR</i> (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Because the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named <i>walb</i>. We cloned other <i>walb</i> copies residing in the genomes of this species and another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of <i>gag</i>, <i>pol</i>, and <i>env</i> of retroviruses. It is not known through which of the following likely processes the walb copy was inserted into <i>TYR</i>: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into <i>TYR</i> is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances.

The Efficiency of Long Primers Compared to The Short Primer for RAPD Technique in Date Palm

Random Amplified Polymorphic DNA (RAPD) applies single arbitrary short primers (8-12nucleotides) to produce many amplified discrete DNA. Limited reports and studies were done onthe use of long primers (over 12 bases). This study was performed to investigate the potential valueof long primers (15-21 bases) for generating RAPD polymorphisms. We compared both short andlong primers in RAPD assays of two date palm cultivars grown in Malaysia: Ajwa and Barhi. Thenumber of produced polymorphic fragments ranged in order from 2 and 38 bands for short andlong primers in Ajwa. Meanwhile, more polymorphic fragments were generated by long primersin Barhi, which were 50 and only five bands for short primers. 18-mer GY107 and 20-mer CO4primers yielded 100% polymorphism in Ajwa and Barhi, respectively. Moreover, long primersproduced more DNA fragments and a wider range of DNA fragment sizes (from 140-1600 bp,with respect to 300-1000 bp obtained with 10-mer primers). Hence, a significant correlation wasobserved between primer length and the number of polymorphic fragments within the long primergroup, suggesting that increasing primer length above 15 bases may demonstrate enhancedproduction of more polymorphism.

GHR/HindIII Locus Polymorphisms in Intron-2 GHR Gene of Papua Local Chicken

This study aimed to detect single nucleotide polymorphisms (SNPs) in intron-2 on growth hormone receptor (GHR) gene in Papua local chickens using the PCR-RFLP method to study its relationship with growth characteristics. Data on the bodyweight of 49 chickens aged 1, 2, 3, and 4 months (22 males, 27 females) and DNA samples were used for this study. The DNA fragment of size 718 bp in intron-2 of the GHR gene from the study chicken was successfully amplified using a pair of specific primers. The PCR-RFLP/HindIII analysis results found this locus's two genotypes (HindIII++ and HindIII--). HindIII+ and HindIII- alleles were 0.02 and 0.98, respectively.

DNA-FACE™ - An Escherichia coli-based DNA Amplification-Expression Technology for Automatic Assembly of Concatemeric ORFs and Proteins

DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal biotechnological platform, developed as Escherichia coli (E. coli) system. It is based on the ordered, head-to-tail directional ligation of the amplified DNA fragments. The technology enables the construction of targeted biomolecules - genetically programmed, concatemeric DNA, RNA, and proteins, designed to fit a particular task. The constructed, “artificial” (never seen in Nature) tandem repeat macromolecules, with specialized functions, may contain up to 500 copies of monomeric units. The technology greatly exceeds the current capabilities of chemical gene synthesis. The vector-enzymatic DNA fragment amplification assembles the DNA segments, forming continuous Open Reading Frames (ORFs). The obtained ORFs are ready for high-level expression in E. coli without a need for subcloning. The presented method has potential applications in pharmaceutical industry and tissue engineering, including vaccines, biological drugs, drug delivery systems, mass-production of peptide-derived biomaterials, industrial and environmental processes. The technology has been patented worldwide and used successfully in the construction of anti-HBV vaccines, pro-regenerative biological drugs and, recently, the anti-SARS-CoV-2 vaccine. The anti-SARS-CoV-2 vaccine, developed using the DNA-FACE™ technology, is nontoxic and induces strong immunological response to recombinant human spike and nucleocapsid proteins, as shown in animal studies.

First Report of ‘Candidatus phytoplasma ziziphi’ in Sweet Potato in China

Sweet potato (Ipomoea batatas; family Convolvulaceae) is an important food crop and serves as a ground cover in orchards of jujube trees. In August 2020, sweet potatoes growing under jujube trees showed obvious jujube witches’ broom phytoplasma disease symptoms in an abandoned jujube orchards in Xinzheng county, Henan province, China (GPS：113°49′56″N， 34°35′54″E). The sweet potato plants were showing symptoms such as small, yellowing leaves and witches’ broom. (Figure 1). The incidence of symptomatic sweet potato plants in the orchard (60× 100 m2 ) was about 60%. Leaf samples of sweet potatoes and jujube trees exhibiting disease symptoms were collected to confirm the presence of JWB phytoplasma by PCR. The phytoplasma primers R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) were used. Leaf samples of three sweet potato plants and three jujube trees exhibiting disease symptoms were collected to confirm the presence of JWB phytoplasma by PCR. Two healthy sweet potatoes collected from the experimental station at Henan Agricultural University and two healthy, in vitro-grown jujube plantlets were used as negative controls. A JWB-diseased in vitro-grown jujube plantlets was set as the positive control. The total DNA of 3 leaves from each sample was extracted using the Hi-DNA secure Plant Kit (DP350, TianGen, Beijing, China). The phytoplasma amplicon was detected in all leaf samples (Figure 2). The expected 16S rDNA (GenBank accession number MW990090) amplicon is 1,348 bp, and the expected secA (GenBank accession number MZ292648) amplicon is 842 bp. These two fragments were detected in the sweet potato samples (Figure 2a, 2b). The DNA fragment amplified from the diseased sweet potato and the diseased jujube leave samples exhibiting disease symptoms using the 16S rRNA primers was then sequenced (Sangon Biotech, Shanghai, China) and was 100% identical to that of 23 JWB phytoplasma strains through BLAST analysis. The DNA fragment amplified using the secA primers was sequenced and was 100% identical to that of JWB phytoplasma strain ZQ secA gene (GU471770.1). RFLP analysis of the 16S rRNA sequence fragment by the online tool iPhyClassifier (Wei et al., 2007) indicated that the pathogen strain was a member of subgroup 16SrV-B and a ‘Candidatus Phytoplasma ziziphi’-related strain. Sweet potato witches’ broom disease was reported by Gundersen (1994) and Lee, I. M. (2004). Sweet potato little leaf disease was reported and classified as a phytoplasma in the 16 SrII group (Tairo et al., 2006). According to the evolutionary analysis, the 16S rRNA nucleotide sequences found in sweet potato in this study were quite different from that detected in sweet potato little leaf disease (Figure 2c). Taken together, the results indicated that the phytoplasma associated with sweet potato in the present study is a ‘Ca. Phytoplasma ziziphi’ strain. To our knowledge, this is the first report of its presence in sweet potato in China.

Polymorphism of Insulin-induced gene 1 (INSIG1) of three local beef cattle in Indonesia

Insulin-induced gene 1 (INSIG1) encoded protein that blocked of proteolysis activity from protein sterol regulatory element binding proteins (SREBP). The role gene plays of cholesterol, fatty acid, and glucose metabolism. Indonesia has many several beef cattle which has good quality of growth trait. The aim of this study was to identify polymorphism of INSIG1 gene (SNP 4366 (A>G) and 4534 (T>C)) of three local beef cattle in Indonesia (Bali, Pasundan and Ongole Decendent). One hundred and twenty seven samples were used in this study consisting of Bali cattle (46) from Pulukan Bali Island, Pasundan (36) from UPTD BPPT Beef cattle Ciamis West Java and Ongole Grade (OD) (45). DNA was extracted from whole blood using High Salt method then genotyping used PCR-RFLP method with RsaI and TaqI enzymes. Parameters in this study were genotype and allele frequencies, heterozigosity observed (Ho), expected (He), Hardy-Weinberg Equilibrium (HWE) and Polymorphism Information Content (PIC). Result showed that a 428 bp of DNA fragment was successfully amplified and digested. Three variant of genotypes with two alleles were identified. For SNP 4366 (A>G) were AA, AG and GG with dominant of G allele while SNP 4534 (T>C) were CC, CT and TT with dominant C allele. Both of SNPs in each of breed populations was in Hardy-Weinberg Equilibrium and polymorphic. Additionally, PIC value reached moderate. In conclusion, polymorphism was found in beef cattle and it could be early study for genetic diversity

Polymer Chain Reaction (PCR): Principle and Applications

The new, standard molecular biologic system for duplicating DNA enzymatically devoid of employing a living organism, like E. coli or yeast, represents polymerases chain reaction (PCR). This technology allows an exponential intensification of a minor quantity of DNA molecule several times. Analysis can be straightforward with more DNA available. A thermal heat cycler performs a polymerization chain reaction that involves repeated cycles of heating and cooling the reactant tubes at the desired temperature for each reaction step. A heated deck is positioned on the upper reaction tube to avoid evaporating the reaction mixture (normally volumes range from 15 to 100 l per tube), or an oil layer can be placed on a reaction mixture surface. The amplified DNA fragment is determined based on selecting primers in addition to the starting and end of the DNA fragment. The primers stand for short, artificial DNA stripes, no higher than fifty (typically 18-25bp) nucleotides have been based on a starting and ending of DNA fragment to be amplified. DNA-polymerase connects and starts a new DNA strand synthesis The PCR products can be visualized by dual foremost methods: (1) staining of the product of DNA amplified by a chemical dye like bromide ethidium, or (2) marking of fluorescent dyes (fluorophores) PCR primers or nucleotides before amplification of PCRs. PCR offers some benefits. First, it is a simple method of understanding and using and quick results. It has an extremely sensitive technology with the potential for sequencing, cloning, and analyzing millions or milliards of copies of a particular product.