DNA Sequence Homology Research Articles

Genotyping single nucleotide polymorphisms in homologous regions using multiplex kb level amplicon capture sequencing.

Single nucleotide polymorphisms (SNPs) in homologous regions play a critical role in the field of genetics. However, genotyping these SNPs is challenging due to the presence of repetitive sequences within genome, which demand specific method. We introduce a new, mid-throughput method that simplifies SNP genotyping in homologous DNA sequences by utilizing a combination of multiplex kb level PCR (PCR size 2.5k-3.5kb) for capturing targeted regions and multiplex nested PCR library construction for next-generation sequencing (Multi-kb level capture-seq). First of all, we randomly selected 7 SNPs in homologous regions and successfully captured 6-plex kb level amplicons (one of segments contains 2 SNPs, while the remaining segments each have only one SNP) in a single tube. And then, the amplification products were subjected to multiplex nested PCR for library construction and sequenced on Illumina platform. We tested this strategy using 600 amplicons from 100 samples and accurately genotyped 96.8% of target SNPs with a coverage depth of ≥ 15×. For the uniformity within the samples, over 66.7% (4/6) of the amplicons had a coverage depth above 0.2-fold of average sequencing depth. To validate the accuracy of this approach, we performed Ligase detection reaction PCR for genotyping the 100 samples, and found that the genotyping data was 97.71% consistent with our NGS results. In conclusion, we have developed a highly efficient and accurate method for SNP genotyping in homologous regions, which offers researchers a new strategy to explore the complex regions of genome.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Nonlocal structural effects of water on DNA homology recognition

The mechanism behind mutual recognition of homologous DNA sequences prior to genetic recombination is one of the remaining puzzles in molecular biology. Leading models of homology recognition, based on classical electrostatics, neglect the short-range nonlocal screening effects arising from structured water around DNA, and hence may only provide insight for relatively large separations between interacting DNAs. We elucidate the role of the effects of the nonlocal dielectric response of water on DNA–DNA interaction and show that these can dramatically enhance the driving force for recognition.

Water structural effects on DNA–DNA interactions and homologous recognition

Comprehending the principles underlying the interaction between DNA molecules in electrolyte solution is of crucial importance for understanding the phenomena of DNA condensation, recognition of homologous genes, and properties of DNA liquid crystals. Although DNA was considered in all its helical complexity over the last two decades, only a primitive, local, electrostatic model of the aqueous solution was considered. The forces acting between DNA molecules however must be influenced by the structure of the liquid separating them. In this paper we make a step towards unravelling earlier neglected effects of water structure on DNA–DNA interactions. We develop a model of DNA interaction accounting for the nonlocal dielectric response of water and of the electrolyte plasma dissolved in it. We focus particularly on the study of the dipolar overscreening effect, which leads to decaying oscillations of the electrostatic potential about DNA, and how it will affect electrostatic energy surfaces as a function of interaxial separation and mutual azimuthal orientation of the parallelly aligned interacting DNAs. We found that going beyond the classical electrostatic description of water (the so-called primitive model) reveals a complex oscillatory interaction surface in space, with many possible metastable configurations. These however vanish rapidly with smearing of the DNA charge distribution, due to dephasing of the oscillatory components of the interaction. This dephasing results in the suppression of oscillation amplitudes and leads to the dominance of non-oscillatory components in spatial dipolar correlations and the Debye screening. These dominant correlation patterns are shown to lead to a substantial enhancement of the difference between the interaction of homologous and nonhomologous DNA sequences, deepening and sharpening the homology recognition well.

Identification of phytase producing bacteria from acidifying Tithonia diversifolia: Potential for ruminant feed development

Phytate content in feed ingredients can negatively impact digestibility and palatability. To address this issue, it is necessary to study microbes capable of breaking down phytate content. This study aimed to isolate and characterize phytase-producing bacteria from decaying materials rich in phytic acid. The research was conducted in several stages. The first stage involved isolating phytase-producing bacteria from the acidification of Tithonia diversifolia using growth media containing Na-phytate. Bacterial isolates that produced clear zones were then tested for their activity and ability to produce several enzymes, specifically phytase, cellulase, and protease. The next step was to test the morphological characteristics of the bacterial isolate. The final stage of bacterial identification consisted of DNA isolation, followed by PCR amplification of the 16S rRNA gene, DNA sequence homology analysis, and construction of a phylogenetic tree. Based on research, three isolates were found to produce clear phytase zones: isolates R5 (20.3 mm), R7 (16.1 mm) and R8 (31.7 mm). All isolates were able to produce the enzymes phytase (5.45–6.54 U/ml), cellulase (2.60–2.92 U/ml), and protease (22.2–23.4 U/ml). Metagenomic testing identified isolate R7 and R8 as Alcaligenes faecalis and isolate R5 as Achromobacter xylosoxidans. The isolation and characterization of phytase-producing bacteria from Tithonia diversifolia acidification resulted in the identification of two promising candidates that can be applied as sources of phytase producers. Phytase-producing bacteria can be utilized to improve digestibility and palatability in animal feed.

A new species of the genus Leptobrachella Smith 1925 (Amphibia, Anura, Megophryidae) from Guangxi, China.

A new species of Leptobrachella, L.wumingensissp. nov., was described from the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China based on morphological, molecular and bioacoustic data. Phylogenetic analysis of 16S mtDNA fragments revealed that the new species is closely related to L.damingshanensis. Uncorrected p-distances between the new species and all homologous DNA sequences available for the 16S gene of Leptobrachella are greater than 7.1%. Morphologically, L.wumingensissp. nov. differs from its congeners in several ways, including a medium body size (SVL 26.0-26.7 mm in males, 30.6-34.8 mm in females), lack of toe webbing and lateral fringes, shagreened and granular dorsal surface, pale brown dorsum with darker brown markings, iris bicolored, with the upper half copper and fading to silver in the lower half, and the presence of small irregular black spots and tangerine tubercles on the flanks. Furthermore, we found the new species to have two types of advertisement calls and relatively high dominant frequencies, making it distinct from its congeners.

In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search.

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks (DSB). HR uses an undamaged homologous DNA sequence as a template for copying the missing information, which requires identifying a homologous sequence among megabases of DNA within the crowded nucleus. In eukaryotes, the conserved Rad51-single-stranded DNA nucleoprotein filament (NPF) performs this homology search. Although NPFs have been extensively studied in vitro by molecular and genetic approaches, their in vivo formation and dynamics could not thus far be assessed due to the lack of functional tagged versions of Rad51. Here we develop and characterize in budding yeast the first fully functional, tagged version of Rad51. Following induction of a unique DSB, we observe Rad51-ssDNA forming exceedingly long filaments, spanning the whole nucleus and eventually contacting the donor sequence. Emerging filaments adopt a variety of shapes not seen in vitro and are modulated by Rad54 and Srs2, shedding new light on the function of these factors. The filaments are also dynamic, undergoing rounds of compaction and extension. Our biophysical models demonstrate that formation of extended filaments, and particularly their compaction-extension dynamics, constitute a robust search strategy, allowing DSB to rapidly explore the nuclear volume and thus enable efficient HR.

RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens.

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

Technical feasibility of newborn screening for spinal muscular atrophy by next-generation DNA sequencing.

Newborn screening (NBS) assays for spinal muscular atrophy (SMA) typically use a polymerase chain reaction (PCR) based assay to identify individuals with homozygous deletion in exon 7 of the SMN1 gene. Due to high DNA sequence homology between SMN1 and SMN2, it has previously been difficult to accurately bioinformatically map short reads from next-generation DNA sequencing (NGS) to SMN1, resulting in low analytical performance and preventing NGS being used for SMA screening. Advances in bioinformatics have allowed NGS to be used in diagnostic settings, but to date these assays have not reached the scale required for high volume population newborn screening and have not been performed on the dried blood spot samples that NBS programs currently use. Here we integrate an NGS assay using hybridisation-based capture with a customised bioinformatics algorithm and purpose designed high throughput reporting software into an existing NBS program to achieve a laboratory workflow for population SMA screening. We tested the NGS assay on over 2500 newborns born over 2weeks in a NBS program in a technical feasibility study and show high sensitivity and specificity. Our results suggest NGS may be an alternate method for SMA screening by NBS programs, providing a multiplex testing platform on which potentially hundreds of inherited conditions could be simultaneously tested.

Rad52’s DNA annealing activity drives template switching associated with restarted DNA replication

It is thought that many of the simple and complex genomic rearrangements associated with congenital diseases and cancers stem from mistakes made during the restart of collapsed replication forks by recombination enzymes. It is hypothesised that this recombination-mediated restart process transitions from a relatively accurate initiation phase to a less accurate elongation phase characterised by extensive template switching between homologous, homeologous and microhomologous DNA sequences. Using an experimental system in fission yeast, where fork collapse is triggered by a site-specific replication barrier, we show that ectopic recombination, associated with the initiation of recombination-dependent replication (RDR), is driven mainly by the Rad51 recombinase, whereas template switching, during the elongation phase of RDR, relies more on DNA annealing by Rad52. This finding provides both evidence and a mechanistic basis for the transition hypothesis.

Genome Protection by DNA Polymerase θ.

DNA polymerase θ (Pol θ) is a DNA repair enzyme widely conserved in animals and plants. Pol θ uses short DNA sequence homologies to initiate repair of double-strand breaks by theta-mediated end joining. The DNA polymerase domain of Pol θ is at the C terminus and is connected to an N-terminal DNA helicase-like domain by a central linker. Pol θ is crucial for maintenance of damaged genomes during development, protects DNA against extensive deletions, and limits loss of heterozygosity. The cost of using Pol θ for genome protection is that a few nucleotides are usually deleted or added at the repair site. Inactivation of Pol θ often enhances the sensitivity of cells to DNA strand-breaking chemicals and radiation. Since some homologous recombination-defective cancers depend on Pol θ for growth, inhibitors of Pol θ may be useful in treating such tumors.

Molecular phylogeny of Annonaceae species from Binh Chau-Phuoc Buu Nature Reserve based on two chloroplast gene regions

In this study, based on molecular biology techniques, the matK and trnL-F regions of ten Annonaceae species grown in Binh Chau-Phuoc Buu Nature Reserve, Vietnam were successfully amplified and sequenced for the first time. Along with other highly homologous DNA sequences from the GenBank database, the molecular phylogeny of ten studied species was also established. By using the alignment tool of NCBI database, the percentage of identity among sequences of studied species was also presented. The study aims to partially contribute to the further understanding of the evolutionary relationships among Annonaceae species.

Abstract 5688: RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers

Abstract Genome instability has long been considered the primary driver of most cancer types. A double strand break (DSB) in DNA can have deleterious consequences for a cell, which if not repaired faithfully, can lead to mutations and chromosomal rearrangements, or even cell death. DSBs can be processed by several DNA repair pathways, of which homologous recombination (HR) is the preferred method due to its error-free nature. HR uses an intact homologous DNA sequence as a template for recovering the information lost at the break site. A significant proportion of all cancers, especially triple-negative breast, ovarian pancreatic and prostate cancers, have loss of function alterations affecting genes involved in HR-mediated DNA repair. Alternate repair pathways operate when HR is defective in tumors, but the pathways operative in this context remain a matter of contention. Previous work in vivo in yeast and in vitro systems has established a new role of RNA in DNA repair. Owing to its abundance in the cell and its sequence similarity to parental DNA, we sought to define whether RNA can act as a template for the repair of DSBs in human cells. We developed a novel high throughput assay to test if DNA breaks can be repaired using RNA as an alternative template in mammalian cells. Human cells were transfected with a guide RNA cloned in a Cas9 expression vector to generate a site-specific DSB at the AAVS1 locus, a safe harbour, in the human genome. Furthermore, a donor template in the form of DNA or RNA (homologous to the DSB locus) containing a unique mutational signature was provided at the time of transfection. The unique mutational signature enables us to determine if the donor has been utilized as a template for DNA repair. Using this assay, we demonstrate that cells can use a spliced RNA transcript as a functional template to repair a DSB. We have identified that Rev3L, a key component of the translesion synthesis polymerase Pol Zeta (ζ), has a novel reverse-transcriptase activity in human cells and can help repair the DSB using RNA as a template. Further characterization of this repair pathway and its associated mutational scar will provide new insights into the mutational signatures seen in HR-defective cancers, enabling a better understanding of the DNA repair pathways upregulated in these tumours. The proposed studies could help prioritize novel therapeutic approaches by exploiting synthetic lethality in HR-deficient cancers as well as HR-proficient cancers when used in combinatorial cancer therapy. Citation Format: Manisha Jalan, Juber Patel, Kyrie S Olsen, Sana Ahmed-Seghir, Daniel S Higginson, Jorge S Reis-Filho, Nadeem Riaz, Simon N Powell. RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5688.

Bioprospecting and Molecular Identification of Used Transformer Oil-Degrading Bacteria for Bioplastics Production

One of the major impediments to the commercialization of biodegradable plastic is the high cost of substrate. Consequently, there is a continuous search for effective microorganisms and cheaper carbon substrates to reduce the high production cost. In this study, waste transformer oil-degrading bacteria were isolated from soil, wastewater, and sediment samples, using a mineral salt medium (MSM) supplemented with 1% waste transformer oil as the sole carbon source. The isolates were screened for polyhydroxyalkanoates (PHA) production using Nile red staining and fluorescence microscopy. PHA granules accumulation was confirmed using transmission electron microscopy. Oil degradation analysis was accomplished using solvent extraction and gravimetric methods whereas, the bacteria were identified using 16S DNA sequence homology. A total of 62 transformer oil-degrading bacteria were isolated, out of which 16 (26%) showed positive results for Nile red fluorescence microscopy. The identified organisms belong to four different taxonomic genera of Acinetobacter, Bacillus, Proteus, and Serratia. The percentage of oil degradation observed among the different isolates ranged between 19.58% and 57.51%. Analysis of the PHA extracted from the selected isolate revealed the presence of medium chain length polyhydroxyalkanoates (mcl-PHA). The findings of this work have further highlighted the diversity of the bacteria capable of utilizing waste streams such as waste transformer oil. Consequently, the isolates can be explored as agents of converting waste transformer oil into bioplastics.

ROS1-mediated decrease in DNA methylation and increase in expression of defense genes and stress response genes in Arabidopsis thaliana due to abiotic stresses

BackgroundSmall interfering RNAs (siRNAs) target homologous genomic DNA sequences for cytosine methylation, known as RNA-directed DNA methylation (RdDM), plays an important role in transposon control and regulation of gene expression in plants. Repressor of silencing 1 (ROS1) can negatively regulate the RdDM pathway.ResultsIn this paper, we investigated the molecular mechanisms by which an upstream regulator ACD6 in the salicylic acid (SA) defense pathway, an ABA pathway-related gene ACO3, and GSTF14, an endogenous gene of the glutathione S-transferase superfamily, were induced by various abiotic stresses. The results demonstrated that abiotic stresses, including water deficit, cold, and salt stresses, induced demethylation of the repeats in the promoters of ACD6, ACO3, and GSTF14 and transcriptionally activated their expression. Furthermore, our results revealed that ROS1-mediated DNA demethylation plays an important role in the process of transcriptional activation of ACD6 and GSTF14 when Arabidopsis plants are subjected to cold stress.ConclusionsThis study revealed that ROS1 plays an important role in the molecular mechanisms associated with genes involved in defense pathways in response to abiotic stresses.

Speciation of Genes and Genomes: Conservation of DNA Polymorphism by Barriers to Recombination Raised by Mismatch Repair System

Some basic aspects of human and animal biology and evolution involve the establishment of biological uniqueness of species and individuals within their huge variety. The discrimination among closely related species occurs in their offspring at the level of chromosomal DNA sequence homology, which is required for fertility as the hallmark of species. Biological identification of individuals, i.e., of their biological “self”, occurs at the level of protein sequences presented by the MHC/HLA complex as part of the immune system that discriminates non-self from self. Here, a mechanistic molecular model is presented that can explain how DNA sequence divergence and the activity of key mismatch repair proteins, MutS and MutL, lead to 1) genetic separation of closely related species (sympatric speciation) (Fitch and Ayala, Proceedings of the National Academy of Sciences, 1994, 91, 6717–6720), 2) the stability of genomes riddled by diverged repeated sequences, and 3) conservation of highly polymorphic DNA sequence blocks that constitute the immunological self. All three phenomena involve suppression of recombination between diverged homologies, resulting in prevention of gene sharing between closely related genomes (evolution of new species) as well as sequence sharing between closely related genes within a genome (e.g., evolution of immunoglobulin, MHC, and other gene families bearing conserved polymorphisms).

Immunogenic and Protective Features of the Recombinant Vaccinia Virus Strain Expressing Cassette of Genes of Marburg Virus Structural Proteins

The aim of the study was to create a highly immunogenic vaccine construct based on a recombinant variant of a replication-defective MVA strain of vaccinia virus, expressing virus-like particles that mimic natural infection with Marburg virus. Materials and methods. The recombinant virus was obtained through recombination between homologous viral DNA sequences and the insertion plasmid pDel2-GP-VP-Pat which carries transgenes of the structural proteins GP and VP40 of Marburg virus, flanked by fragments of MVA strain genome. Structure of the recombinant virus was confirmed in PCR and using sequencing, transgenes expression was analyzed by Western blotting, viruslike particles formation was recorded using electron microscopy. Evaluation of immunogenicity and protectivity was carried out using a guinea pig model. The antibody titer was determined in enzyme-linked immunosorbent assay. To assess T-cell response, the intracellular staining of cytokines was used, followed by analysis of samples on a flow cytometer. Results and discussion. On the basis of highly attenuated MVA strain of vaccinia virus a recombinant variant MVA-GP-VP40-MARV has been constructed, carrying a cassette of transgenes, GP and VP40, of Marburg virus in the region of deletion II of the genome. The expression of transgenes in MVA-permissive CER cells infected with recombinant MVA-GP-VP40-MARV strain and secretion of GP and VP40 proteins into culture medium have been demonstrated. Electron microscopy analysis has revealed the presence of Marburg virus-like particles in the culture medium of cells 12 hours after infection. Double vaccination of guinea pigs with MVA-GP-VP40-MARV strain at a dose of 108 PFU/animal induced the formation of antibodies to Marburg and vaccinia viruses, as well as 100 % protection against lethal Marburg virus infection (50 LD50). Using original TEpredict software, the structure of T-helper epitopes of GP protein has been predicted. Using the ICS method, the biological activity of these epitopes has been experimentally confirmed and it was shown that they provide the induction of a T-cell immune response as part of the MVA-GP-VP40-MARV vaccine construct.

Antimicrobial resistance and virulence in Helicobacter pylori: Genomic insights

Microbes evolve rapidly by modifying their genome through mutations or acquisition of genetic elements. Antimicrobial resistance in Helicobacter pylori is increasingly prevalent in India. However, limited information is available about the genome of resistant H. pylori isolated from India. Our pan- and core-genome based analyses of 54 Indian H. pylori strains revealed plasticity of its genome. H. pylori is highly heterogenous both in terms of the genomic content and DNA sequence homology of ARGs and virulence factors. We observed that the H. pylori strains are clustered according to their geographical locations. The presence of point mutations in the ARGs and absence of acquired genetic elements linked with ARGs suggest target modifications are the primary mechanism of its antibiotic resistance. The findings of the present study would help in better understanding the emergence of drug-resistant H. pylori and controlling gastric disorders by advancing clinical guidance on selected treatment regimens.

Nucleosome-induced homology recognition in chromatin.

One of the least understood properties of chromatin is the ability of its similar regions to recognize each other through weak interactions. Theories based on electrostatic interactions between helical macromolecules suggest that the ability to recognize sequence homology is an innate property of the non-ideal helical structure of DNA. However, this theory does not account for the nucleosomal packing of DNA. Can homologous DNA sequences recognize each other while wrapped up in the nucleosomes? Can structural homology arise at the level of nucleosome arrays? Here, we present a theoretical model for the recognition potential well between chromatin fibres sliding against each other. This well is different from the one predicted for bare DNA; the minima in energy do not correspond to literal juxtaposition, but are shifted by approximately half the nucleosome repeat length. The presence of this potential well suggests that nucleosome positioning may induce mutual sequence recognition between chromatin fibres and facilitate the formation of chromatin nanodomains. This has implications for nucleosome arrays enclosed between CTCF-cohesin boundaries, which may form stiffer stem-like structures instead of flexible entropically favourable loops. We also consider switches between chromatin states, e.g. through acetylation/deacetylation of histones, and discuss nucleosome-induced recognition as a precursory stage of genetic recombination.

12. Isolate Of Bacteriua Cellulase Enzyme Production In The Rumen Of Aceh Cattle Base On Analysis Homology 16S rRNA

The rumen of cattle is well known one of a bacterium cellulolitic production that have ability to degrade cellulose for produce cellulase enzymes. The aims of the present study is to determine the type of bacteria produced cellulase enzymes in the rumen of cattle aceh. The diversity of cellulolitic bacteria is determined by analysis of 16S rRNA genes. Isolation of cellulolytic bacteria, total DNA isolation, agarose gel electrophoresis, amplification of the 16S rRNA gene, determining the DNA sequence, DNA sequence homology and alignment of DNA sequences. Isolation of bacterium cellulolitic, isolating total DNA, gel electroforesis agarosa, amplified of gen 16S rRNA, determination the DNA barcode, homology DNA barcode, and alignment of DNA sequences. The sample of rumen liquid was taked from RPH , then maked isolation of total DNA to get pure DNA from bacterium whos result and amplification DNA of bacterium cellulolitic separated by electroforesis gel agarosa. Gene of bacterium cellulolitic was isolated based Polymerase Chain Reaction methode. Alignment is done by using BlastN program, then use CLustalW program. It can be concluded that the species of bacteria (FKH_USK_6) in rumen has a closeness with Coronabacter sakazakii bacteria strain PNP8 of 97% according to the sizeof the DNA sequence and the same position on an existing gene in GenBank.