Category Archives: DNA

University of Oxford researchers have contributed to a study which used ancient DNA to identify the earliest known people to have suffered from various genetic disorders affecting the number of sex chromosomes. The findings have been published today in Communications Biology.

Aneuploidy occurs when a persons cells have an extra or missing chromosome. If this occurs in the sex chromosomes, this can cause differences such as delayed development or changes in height around puberty.

Led by the Francis Crick Institute, the research team developed a new technique to measure the number of chromosomes in ancient genomes more precisely. In this way, they identified:

To overcome this, the team developed a computational method which counts the number of copies of X and Y chromosomes and compares the outcome to a predicted baseline (what one would expect to see).

The team used the new method to analyse ancient DNA from a large dataset of individuals collected as part of the Thousand Ancient British Genomes project across British history, identifying six individuals with aneuploidies across five sites in Somerset, Yorkshire, Oxford, and Lincoln. The individuals lived across a range of time periods, from the Iron Age (2500 years ago) up to the Post-Medieval Period (about 250 years ago).

They identified five people who had sex chromosomes which fell outside of the XX or XY categories. All were buried according to their societys customs although no possessions were found with them to shed more light on their lives.

By investigating details on the bones, the research team could see that it was unlikely that the individual with Turner syndrome had gone through puberty and started menstruation, despite their estimated age of 18-22. Their syndrome was shown to be mosaic: some cells had one copy of chromosome X and some had two.

Co-author of the study Rick Schulting, Professor of Scientific and Prehistoric Archaeology at the University of Oxford, said: The results of this study open up exciting new possibilities for the study of sex in the past, moving beyond binary categories in a way that would be impossible without the advances being made in ancient DNA analysis.

The study Detection of chromosomal aneuploidy in ancient genomes has been published inCommunications Biology.

The study was led by the Francis Crick Institute and involved archaeologists from the University of Oxford, the Wells and Mendip Museum, University of York, University of Bradford, Oxford Archaeology, York Osteoarchaeology, and Network Archaeology. It was supported by Lincolnshire County Council, Magdalen College, and Balfour Beatty for National Highways.

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New technique identifies earliest cases of genetic disorders from ancient DNA - University of Oxford

Researchers trying to find better drugs for MS now have a better idea of what theyre up against when it comes to certain autoimmune diseases. As explained by Lars Fugger, a neurologist at the University of Oxford who was involved in the study, scientists are trying to undo 5,000 years of evolutionary honing.

Unfortunately, the research doesnt offer a roadmap for how to design new and better drugs. But Lugger suggests that the work might at least point to a better way. Current MS therapies generally work by damping down the immune system, but given that these mutations arose to protect us, perhaps the better approach would be to recalibrate immune cells activity rather than shutting them down altogether.

Ideally, with these data in hand and eventually made available to other researchers an exploration other health conditions will unfold. The signal of risk wont always be as clear and meaningful as with MS, but there are plenty of diseases where this type of data can be used to solidify hypotheses and illuminate biology.

As that work evolves, the field must do better a job of expanding the study of ancient (and modern) DNA beyond Europe and North America. Although all humans are genetically very similar, our evolutionary pasts might differ and, as this MS paper nicely highlights, those histories can affect our understanding of current health issues.

Some of the focus on European ancestors is practical. Ancient DNA is better preserved in colder, drier climates, making it harder to extract useful genetic information from samples in, for example, the tropics. But researchers say funding for projects in less-represented populations has lagged significantly.

The problem is not limited to ancient DNA. Much of the work in modern genetics has been devoted to studying people outside of North America and Europe. Asgari points out that 80 per cent of large genetic studies focus on populations of European ancestry, yet that group makes up just 18 per cent of the global population.

While the field has become far better at acknowledging these knowledge and resource gaps, more needs to be done to close them. Each region has diseases that might be better understood by comparing ancient and modern DNA. Asgari points to sickle cell disease, which is believed to have arisen as a protection against malaria, or the high prevalence of diabetes in the Middle East, as areas that deserve the same kind of study.

Theres still so much to learn about the human genome. Well learn more, faster, if we cast a wider net.

Disclaimer: The views expressed above are the author's own. They do not necessarily reflect the views of DH.

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Ancient DNA could be hiding all kinds of health secrets - Deccan Herald

Researchers have created the worlds largest ancient human gene bank by analysing the bones and teeth of almost 5,000 humans who lived across western Europe and Asia up to 34,000 years ago.

By sequencing ancient human DNA and comparing it to modern-day samples, the international team of experts mapped the historical spread of genes and diseases over time as populations migrated.

The astounding results have been revealed in four trailblazing papers published in the journal Nature and provide new biological understanding of debilitating disorders.

The study involved a large international team led byProfessor Eske Willerslevat the Universities of Cambridge and Copenhagen,Professor Thomas Wergeat the University of Copenhagen, andProfessor Rasmus Nielsenat University of California, Berkeley, with contributions from 175 researchers from around the globe. They found:

Future analysis is hoped to reveal more about the genetic markers of autism, ADHD, schizophrenia, bipolar disorder, and depression.

Northern Europe has the highest prevalence of multiple sclerosis in the world.

The new study foundthe genes that significantly increase a persons risk of developing multiple sclerosis (MS) were introduced into north-western Europe around 5,000 years ago by sheep and cattle herders migrating from the east.

By analysing the DNA of ancient human bones and teeth, found at documented locations across Eurasia, researchers traced the geographical spread of MS from its origins on the Pontic Steppe (a region spanning parts of what are now Ukraine, South-West Russia and the West Kazakhstan Region).

They found that the genetic variants associated with a risk of developing MS travelled with the Yamnaya people livestock herders who migrated over the Pontic Steppe into North-Western Europe.

These genetic variants provided a survival advantage to the Yamnaya people, most likely by protecting them from catching infections from their sheep and cattle. But they also increased the risk of developing MS.

It must have been a distinct advantage for the Yamnaya people to carry the MS risk genes, even after arriving in Europe, despite the fact that these genes undeniably increased their risk of developing MS. These results change our view of the causes of multiple sclerosis and have implications for the way it is treated.

Professor Eske Willerslev, jointly at the Universities of Cambridge and Copenhagen, Fellow of St Johns College, expert in analysis of ancient DNA and Director of the project.

The age of specimens ranges from the Mesolithic and Neolithic through the Bronze Age, Iron Age and Viking period into the Middle Ages. The oldest genome in the data set is from an individual who lived approximately 34,000years ago.

The findings provide an explanation for the North-South Gradient, in which there are around twice as many modern-day cases of MS in northern Europe than southern Europe, which has long been a mystery to researchers.

From a genetic perspective, the Yamnaya people are thought to be the ancestors of the present-day inhabitants of much of North-Western Europe. Their genetic influence on todays population of southern Europe is much weaker.

Previous studies have identified 233 genetic variants that increase the risk of developing MS. These variants, also affected by environmental and lifestyle factors, increase disease risk by around 30 percent. The new research found that this modern-day genetic risk profile for MS is also present in bones and teeth that are thousands of years old.

These results astounded us all. They provide a huge leap forward in our understanding of the evolution of MS and other autoimmune diseases. Showing how the lifestyles of our ancestors impacted modern disease risk just highlights how much we are the recipients of ancient immune systems in a modern world.

Dr William Barrie, postdoc in the University of Cambridges Department of Zoology and first author of the MS study.

Multiple sclerosis is a neurodegenerative disease in which the bodys immune system mistakenly attacks the insulation surrounding the nerve fibres of the brain and spinal cord. This causes symptom flares known as relapses as well as longer-term degeneration, known as progression.

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Ancient DNA reveals reason for high MS and Alzheimer's rates in Europe - ScienceBlog.com

NEW DELHI: Ten years after initiating proceedings on a PIL seeking DNA profiling of all unidentified bodies, the Supreme Court was back at square one as it sought Union government's response afresh on a PIL on the same issue - DNA profiling of unidentified bodies - after being told that a bill on this subject has been withdrawn from Parliament. In 2014, the SC had issued notices to ministry of home affairs, CBI and secretary, department of scientific and industrial research on a PIL by Lokniti Foundation, which had said that absence of a national DNA database is impeding identification of nearly 40,000 bodies that are found across the country every year. After dealing with the PIL for four years, the SC on May 1, 2018 had disposed of the PIL as government had then promised to bring a bill in the monsoon session of Parliament for DNA profiling to enable maintaining records of unidentified and unclaimed bodies or missing persons. Government did introduce a bill on use of DNA technology in 2018 and it was passed by the Lok Sabha. However, it lapsed in 2019 and a new bill on this subject was re-introduced in July 2019. It was sent to the standing committee. During the pendency of the DNA bill, Parliament enacted Criminal Procedure (Identification) Act, 2022 which authorised police and prison authorities to collect biological samples, including DNA, of persons arrested, detained, under-trial or convicted in a criminal case. On July 24 last year, government withdrew from Lok Sabha the DNA Technology (Use and Application) Regulation Bill, 2019, citing the enactment of the 2022 law. But Congress and opposition parties had criticised the move saying the BJP-led NDA government did not want to incorporate the privacy safeguards suggested by the standing committee. On Tuesday, a bench of Chief Justice D Y Chandrachud, and Justices J B Pardiwala and Manoj Misra sought response from government on a PIL filed by advocate Kishan Chand Jain, seeking use of DNA technology to profile unidentified bodies to help relatives identify their near and dear ones who had gone missing. However, before entertaining the PIL, the bench observed that whether to enact a law is completely within the prerogative of Parliament and the courts cannot intervene in this field. To this, Jain said that he was not on the issue of directing the government to move a bill to this effect, but on use of DNA technology to make it easier for relatives to identify a body.

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Supreme Court seeks government's view on DNA profiling of unidentified bodies - IndiaTimes

MOSCOW - According to documents filed this week, Judge John Judge says Bryan Kohberger's defense team will receive some of the DNA records requested from the prosecution.

Kohberger is accused of murder in connection with the stabbing deaths of Ethan Chapin, Madison Mogen, Xana Kernodle, and Kaylee Goncalves at a home near the University of Idaho campus in November of 2022.

"The Court has now completed its review of the information provided by the State and orders the State to discover to the defense a portion of the IGG information. The specific material to be provided is set forth in a sealed order to protect the privacy of the IGG information, including individuals on the family tree," says a court order signed by Judge John Judge on Thursday.

IGG is short for "investigative genetic genealogy." IGG evidence was used to name Kohberger as suspect after he allegedly left a Ka-Bar knife sheath containing DNA evidence at the crime scene.

IGG involves comparing DNA from a crime scene to data from commercial online genealogy services that are often used by consumers to investigate their family tree. Defense attorneys for Kohberger have tried several times to obtain the evidence, but were unsuccessful prior to this week.

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Prosecution Ordered to Release Portion of DNA Evidence to Kohberger's Defense Team - bigcountrynewsconnection.com

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Judge orders more DNA disclosure in University of Idaho murder case - KXLY Spokane

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Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining ... - Nature.com

Ethics statement

Human Study: The lung specimens from the COVID-19 positive human subjects were collected using autopsy (study was IRB Exempt). All donations to this trial were obtained after telephone consent followed by written email confirmation with next of kin/power of attorney per California state law (no in-person visitation could be allowed into the COVID-19 ICU during the pandemic). The detailed patient characteristics were published elsewhere (PMID: 34127431). For normal lung tissues, lung biopsies were obtained after surgical resection of lungs by cardiothoracic surgeons as before [https://elifesciences.org/articles/66417]. Deidentified lung tissues obtained during surgical resection, which were deemed excess by clinical pathologists, were collected using an approved human research protocol (IRB no. 101590). Blood samples were obtained from UTMB Biorepository of research subjects with a laboratory diagnosis of COVID-19 that consented to participate in the Clinical Characterization Protocol for Severe Emerging Infections (UNMC IRB no. 146-20-FB/UTMB IRB no. 20-0066). The normal healthy subjects blood cell pellets were obtained under UTMB IRB no. 14-0131 and 20-0097.

Animal (Hamster) study: Lung samples from 8-week-old male Syrian hamsters were generated from experiments conducted exactly as in previously published studies (PMID: 32540903). Animal studies were approved and performed in accordance with Scripps Research IACUC Protocol no. 20-0003 and UTMB IACUC Protocol no. 2005060.

Publicly available COVID-19 gene expression databases were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus website (GEO)69,70,71. If the dataset was not normalized, RMA (Robust Multichip Average)72,73 was used for microarrays and TPM (Transcripts Per Millions)74,75 was used for RNASeq data for normalization. We used log2 (TPM+1) to compute the final log-reduced expression values for RNASeq data. Accession numbers for these crowd-sourced datasets are provided in the figures and manuscript. Single Cell RNASeq data from GSE145926 was downloaded from GEO in the HDF5 Feature Barcode Matrix Format. The filtered barcode data matrix was processed using Seurat v3 R package76. Pseudo bulk analysis of GSE145926 dataset was performed by adding counts from the different cell subtypes and normalized using log2 (CPM+1). All of the above datasets were processed using the Hegemon data analysis framework77,78,79.

Time (duration in hospital) and status (whether the patient is discharged from hospital) were derived from the hospital-free days post 45-day follow-up from COVID-19 patients (n=100, GSE157103). All non-COVID-19 patients (n=26, GSE157103) were excluded from the analysis. KaplanMeier (KM) analysis is performed using lifelines python package version 0.14.6. All KM analyses use the StepMiner threshold+0.5 noise margin as the threshold to separate the patients into high and low groups.

COVID-19 samples were inactivated by storing in 10 % formalin for 2 days and then were transferred to zinc-formalin solution for another 3 days. The decontaminated tissues were transferred to 70% ethanol and cassettes were prepared for tissue sectioning. The slides containing hamster and human lung tissue sections were de-paraffinized in xylene (Sigma-Aldrich, catalog no. 534056) and rehydrated in graded alcohols to water. For NEIL2 antigen retrieval, slides were immersed in Tris-EDTA buffer (pH 9.0) and boiled for 10min at 100C inside a pressure cooker. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 10min. To block non-specific protein binding 2.5% goat serum (Vector Laboratories, catalog no. MP-7401) was added. Tissues were then incubated with rabbit anti-NEIL2 polyclonal antibody (in house generated, 33) for 1.5h at room temperature in a humidified chamber and then rinsed with TBS or PBS 3x, 5min each. Sections were incubated with horse anti-rabbit IgG (Vector Laboratories, catalog no. MP-7401) secondary antibodies for 30min at room temperature and then washed with TBS or PBS 3x, 5min each; incubated with 3,3-diaminobenzidine tetrahydrochloride (DAB) (Thermo Scientific, catalog no. 34002), counterstained with hematoxylin (Sigma-Aldrich, catalog no. MHS1) for 30s, dehydrated in graded alcohols, cleared in xylene, and cover slipped. Epithelial and stromal components of the lung tissue were identified by staining duplicate slides in parallel with hematoxylin and eosin (Sigma-Aldrich, catalog no. E4009) and visualizing by Leica DM1000 LED (Leica Microsystems, Germany).

IHC images were randomly sampled at different 300300 pixel regions of interest (ROI). The ROIs were analyzed using IHC Profiler80. IHC Profiler uses a spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. The histogram of the DAB intensity was divided into 4 zones: high positive (060), positive (61120), low positive (121180) and negative (181235). High positive, positive, and low positive percentages were combined to compute the final percentage positive for each ROI. The range of values for the percent positive is compared among different experimental groups.

Lung specimens from COVID-19 positive human subjects were collected using autopsy procedures at the University of California San Diego (the study was IRB Exempt) following guidelines from the Centers for Disease Control and Prevention (CDC) and College of American Pathologists autopsy committee. All donations to this trial were obtained after telephone consent followed by written email confirmation with next of kin/power of attorney per California state law (no in-person visitation could be allowed into the COVID-19 ICU during the pandemic). (https://www.cdc.gov/coronavirus/2019-ncov/hcp/guidance-postmortem-specimens.html and https://documents.cap.org/documents/COVID-Autopsy-Statement-05may2020.pdf). Lung specimens were collected in 10 % Zinc-formalin and stored for 72h before processing for histology as done previously81,82.

Blood cell pellets stored in TRIzol LS Reagent (Invitrogen, catalog no. 10296010) were obtained from the UTMB Biorepository for Severe Emerging Infections from research subjects with a laboratory diagnosis of COVID-19 that consented to participate in the Clinical Characterization Protocol for Severe Emerging Infections (UNMC IRB no. 146-20-FB/UTMB IRB no. 20-0066). Samples were used from subjects categorized as having moderate or severe COVID-19 based on the following criteria: moderate disease if requiring oxygen via nasal cannula, severe disease if requiring oxygen via non-invasive ventilation (e.g., CPAP, BiPAP, High-Flow nasal cannula, venturi mask). The normal healthy subjects blood cell pellets were obtained in TRIzol LS Reagent under UTMB IRB # 14-0131 and 20-0097. Total RNA was isolated as per manufacturers protocol and subjected to real time reverse transcriptase-quantitative Polymerase Chain Reaction.

Total RNA extraction was performed from cells using TRIzol Reagent (Invitrogen, catalog no. 15596026) or TRIzol LS Reagent. Total RNA (up to 2g) was used to synthesize cDNA with a PrimeScriptTM RT Kit with gDNA Eraser (TaKaRa, catalog no. RR047A) and qPCR was carried out using TB Green Premix Ex Taq II (Tli RNase H Plus; TaKaRa, catalog no. RR820A) in Applied Biosystems 7500 Real-Time PCR Systems with thermal cycling conditions of 94C for 5min, (94C for 10s, and 60C for 1min) for 40 cycles, and 60C for 5min. The target mRNA levels were normalized to that of GAPDH or 18S RNA. Primer sequences used in the assay are listed in Supplementary Table1. In each case, DNase-treated RNA samples without reverse transcriptase were amplified to test genomic DNA contamination.

Syrian golden hamsters (Hamster/Golden Syrian Hamster/Male/8 weeks old/Charles River/Strain Code 049) experiments were approved by the Scripps Research Institute Institutional Animal Care and Use Committee/Protocol 20-0003, and were carried out in accordance with recommendations. Lung samples were collected from 8-week-old Golden Syrian hamsters post SARS-CoV-2 infection conducted exactly as in a previously published study38. Briefly, lungs from hamsters challenged with SARS-CoV-2 (1106 PFU) were harvested on day 5 (peak weight loss) and NEIL2 protein and mRNA levels were analyzed by IHC and RT-qPCR, respectively. Syrian golden hamsters (Male/8 weeks old) were infected with SARS-CoV2 as approved by the UTMB IACUC (protocol no. 2005060) and nuclear extract was prepared from the uninfected and infected hamster lungs at 10 days post infection as described before33,39, and DNA was extracted from the same samples for LA-qPCR.

A549 cells stably expressing human angiotensin I converting enzyme 2 (A549-ACE2)83 is maintained in Eagles Minimum Essential Media (EMEM; Gibco, Cat # 11095080), containing 10% fetal bovine serum (FBS), 100units/ml penicillin and 100g/mL streptomycin. A549-ACE2 cells grown in six-well plates at ~70% confluence were transduced with recombinant proteins using Pierce Protein Transfection Reagent according to manufacturers recommendations (Pierce, Thermo Scientific, catalog no. 89850). In brief, Pierce reagent (dissolved in 250L of methanol or chloroform) was evaporated to remove traces of solvent and 2g of rNEIL1, or rNEIL2 protein was added in PBS, vortexed, incubated for 5min at room temperature, then the mixture was supplemented with serum free medium. Mixtures were added directly onto the cell monolayers, incubated for 4h in a 5% CO2 containing incubator at 37C and then one volume of 20% serum-containing medium was added for overnight. Transfection efficiency varied between 68 and 75% as determined in parallel experiments by indirect immunofluorescence assays using anti-NEIL2 or anti-NEIL1 (in house generated84) antibodies. Transduced A549/ACE2 cells were infected with SARS-CoV-2 at MOI 11.87. After incubation for an hour with viral inoculum, cells were washed three times with EMEM. Infected cells were harvested at indicated time points in various lysis buffers, depending on the downstream experiment. Supernatants from infected cells were harvested at 24h post-infection for measuring the infectious virus titers by the TCID50 assay using Vero E6 cells. Briefly, 50L supernatants from infected cells were serially diluted (10-fold) in EMEM supplemented with 2% FBS; 100L of serially diluted samples were added to Vero E6 cells grown in 96-well plates and cultivated at 37C for 3 days followed by observation under a microscope for the status of virus-induced formation of cytopathic effect (CPE) in individual wells. The titers were expressed as log TCID50/mL.

Human bronchial epithelium cell line, BEAS-2B (ATCC CRL-9609) stably expressing NEIL2-FLAG,human gastric adenocarcinoma (AGS, ATCC CRL-1739) and human embryonic kidney cells (HEK29385) were grown at 37C and 5% CO2 in DMEM/F-12 (1:1) containing 10% FBS, 100units/ml penicillin and 100units/ml streptomycin. For all experiments, 5060% confluent cells were used. We routinely tested cell lines for mycoplasma contaminations using the PCR-based Venor GeM Mycoplasma Detection Kit (Sigma, catalog no. MP0025). Control or stable BEAS-2B cells at ~70% confluency were transiently transfected with vector expressing GFP with (SARS-CoV2-5-UTR-eGFP construct, synthesized and cloned by GenScript Inc.) or without (UTR-Less-eGFP construct) UTR (100ng) using Lipofectamine TM 2000 (Invitrogen, catalog no. 11668027), according to the suppliers protocol. To monitor transfection efficiency, a reporter gene construct (0.25g) containing -galactosidase downstream to the SV40 promoter was co-transfected. Cells were allowed to recover for 16h in media with serum and then GFP florescence was measured using an ECHO florescent microscope (ECHO Revolve-R4). Total RNA and DNA were isolated for subsequent qPCR analysis.

The proteins in the nuclear extracts (from Hamster lungs)/whole cell extracts A549-ACE2 cells were separated onto a Bio-Rad 420% gradient Bis-Tris gel, then electro-transferred on a nitrocellulose (0.45m pore size; GE Healthcare) membrane using 1X Bio-Rad transfer buffer. The membranes were blocked with 5% w/v skimmed milk in TBST buffer (1X Tris-Buffered Saline, 0.1% Tween 20) and immunoblotted with appropriate antibodies SARS-CoV-2 spike protein (S1-NTD) (Cell Signaling Technology, catalog no. 56996S), GAPDH (BioBharati Life Sciences, catalog no. AB0060), Histidine (BioBharati Life Sciences, catalog no. AB0010), NEIL233, OGG1 (in-house generated86), NEIL1 and APE1 (in-house generated87), and HDAC2 (Histone deacetylase 2, GeneTex, catalog no. GTX109642). The membranes were extensively washed with 1% TBST followed by incubation with anti-isotype secondary antibody (Cell Signaling Technology, catalog no. 7074) conjugated with horseradish peroxidase in 5% skimmed milk at room temperature. Subsequently, the membranes were further washed three times (10min each) in 1% TBST, developed and imaged using kwikquant image analyzer and image analysis software (ver. 5.2) (Kindle Biosciences). Due to cross reactivity of common secondary antibody with the pre developed membrane, the samples were run in parallel gels in similar conditions, and developed with different antibodies. For all the primary antibodies, 1:1000 dilution was used and for secondary antibody, 1:2000 dilution was used.

RNA-ChIP assays were performed as described earlier39. Briefly, cells were cross-linked in 1% formaldehyde for 10min at room temperature. Then 125mM Glycine was added for 5min at room temperature to stop crosslinking and then samples were centrifuged at 1000g at 4C for 5min to pellet the cells. The cell pellet was re-suspended in sonication buffer, containing 50mM Tris-HCl pH 8.0, 10mM EDTA and 1% SDS with 1X Protease inhibitor cocktail and sonicated to an average DNA size of ~300bp using a sonicator (Qsonica Sonicators). The supernatants were diluted with 15mM Tris-HCl pH 8.0, 1.0mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS containing protease inhibitors, and incubated with anti-NEIL1, -NEIL2, -FLAG (Millipore, catalog no. F1804) or normal IgG (Santa Cruz, catalog no. sc-2025) antibodies overnight at 4C. Immunocomplexes (ICs) were captured by Protein A/G PLUS agarose beads (Santa Cruz, catalog no. sc-2003), that were then washed sequentially in buffer I (20mM Tris-HCl pH 8.0, 150mM NaCl, 1mM EDTA, 1% Triton-X-100 and 0.1% SDS); buffer II (same as buffer I, except containing 500mM NaCl); buffer III (1% NP-40, 1% sodium deoxycholate, 10mM Tris-HCl pH 8.0, 1mM EDTA), and finally with 1X Tris-EDTA (pH 8.0) buffer at 4C for 5min each. RNase inhibitor (50Uml1, Roche, catalog no. 03335402001) was added to sonication and IP buffers, and 40Uml1 to each wash buffer. The ICs were extracted from the beads with elution buffer (1% SDS and 100mM NaHCO3) and de-crosslinked for 2h at 65C. RNA isolation was carried out in acidic phenolchloroform followed by ethanol precipitation with GlycoBlue (Life Technologies, catalog no. AM9516) as a carrier. Reverse transcription and cDNA preparation was performed using a PrimeScript RT Kit with gDNA Eraser. RNA-ChIP samples were analyzed by qPCR using specific primers (listed in Supplementary Table1) and represented as percentage input after normalization to IgG.

Wild-type recombinant His-tagged -NEIL2, -NEIL2-ZnF mutant (ZnF-NEIL2mut) and -NEIL1 proteins were purified from E. coli using protocol as described earlier64. Briefly, pET22b (Novagen) vector containing C-terminal 6xHis tagged Coding DNA Sequence (CDS) of various proteins was transformed into E. coli BL21(DE3) RIPL Codon-plus cells (Agilent technologies, catalog no. 230280). The log-phase culture (A600=0.40.6) of E. coli was induced with 0.5mM isopropyl-1-thio--D-galactopyranoside (IPTG) and grown at 16C for 16h. After centrifugation, the cell pellets were suspended in a lysis buffer (Buffer A) containing 25mM Tris-HCl, pH 7.5, 500mM NaCl, 10% glycerol, 1mM -mercaptoethanol (-ME), 0.25% Tween 20, 5mM imidazole, 2mM phenylmethylsulfonyl fluoride (PMSF). After sonication, the lysates were spun down at 13,000rpm and the supernatant was loaded onto HisPur Cobalt Superflow Agarose (Thermo Scientific, catalog no. 25228), previously equilibrated with Buffer A, and incubated for 2h at 4C. After washing with Buffer A with a gradient of increasing concentration of imidazole (10, 20, 30, 40mM), the His-tagged proteins were eluted with an imidazole gradient (80500mM imidazole in buffer containing 25mM Tris-HCl, pH-7.5, 300mM NaCl, 10% glycerol, 1mM -ME, 0.25% Tween 20). After elution, the peak protein fractions (in the range of 100250mM imidazole) were dialyzed against Buffer C (1X PBS, pH 7.5, 1mM dithiothreitol (DTT), and 25% glycerol) and stored at 20C in aliquots.

The Corona virus nsp12 (GenBank: MN908947) gene, cloned into a modified pET24b vector, with the C-terminus possessing a 10 His-tag, was a gift from Dr. Whitney Yin. The plasmid was transformed into E. coli BL21 (DE3) RIPL Codon-plus cells, and the transformed cells were cultured at 37C in LB media containing 100mg/L ampicillin. After the OD600 reached 0.8, the culture was cooled to 16C and supplemented with 0.5mM IPTG. After overnight induction, the cells were harvested through centrifugation, and the pellets were re-suspended in lysis buffer (20mM Tris-HCl, pH 8.0, 150mM NaCl, 4mM MgCl2, 10% glycerol). The rest of the procedure is same as above with following modifications: the His-tagged protein was eluted with an imidazole gradient (80250mM imidazole in buffer containing 20mM Tris-HCl, pH 8.0, 150mM NaCl, 4mM MgCl2, 10% glycerol). Similarly, nsp7 and nsp8 genes, individually cloned in pET22b and pET30a+ vectors, respectively, were expressed in E. coli as described in case of NEIL proteins. After elution, the peak protein fractions of these proteins were dialyzed against Buffer D (20mM Tris-HCl, pH 8.0, 250mM NaCl, 1mM DTT, 25% glycerol) and stored at 20C in aliquots.

For assembling the stable nsp12-nsp7-nsp8 complex, purified nsp12 was incubated with nsp7 and nsp8 at 4C for three hours, at a molar ratio of 1: 2: 2 in a buffer containing 20mM Tris-HCl, pH 7.5, 250mM NaCl and 4mM MgCl288.

RdRp assay for CoV-2-5-UTR ZnF-site was conducted using a self-priming RNA oligo and one short RNA oligo was used as the primer for such assay for CoV-2-3-UTR ZnF-site containing sequence as template (Supplementary Table3). Oligos were mixed at the following final concentrations in 20L reaction volume: Tris-HCl (pH 8, 25mM), RNA short primer (200M), RNA template (2M), [32P]-UTP (0.1M), BSA (1mg/ml), 0.1M GTP, CTP, ATP and 0.01M UTP and SARS-CoV-2 RdRp complex (~0.1M) on ice. For NEIL2 binding, the indicated concentrations of NEIL2 were incubated in the buffer with RNA on ice for 15min. Reactions were stopped after 15, 30 or 60min by the addition of 20L of a formamide/EDTA (50mM) mixture and incubated at 95C for 10min. Samples were run in a 8% urea PAGE using 1x Tris-borate-EDTA as the running buffer. The gels were exposed to a Phosphor screen for 46h and images analyzed using a Typhoon FLA 7000 phosphorimager (GE Healthcare).

RNA-EMSAs with full length CoV-2 5- and 3-UTRs were carried out as previously described89 with some modifications. Briefly, the 297-nt long 5- and the 200-nt long 3-UTR RNAs (sequences in Supplementary Table2, synthesized and cloned in plasmids by GenScript Inc.) were synthesized by in vitro transcription and end-labeled with [-32P] ATP. The indicated concentrations of components were mixed in 15l reactions containing 0.3% poly (vinyl alcohol) (Sigma, catalog no. P-8136), 2mM MgCl2, 0.1 U RNase inhibitor (Biobharati Life Science, India), 1mM DTT, 20mM HEPES-NaOH pH 7.5, 150mM NaCl, and 20% glycerol, and incubated at room temperature for 5min. The RNA-protein complexes were resolved on a native gel (4% 89:1 polyacrylamide gel containing 2.5% glycerol, 50mM Tris, and 50mM glycine) at 4C for 90min. Our EMSAs were designed to examine both the affinity (when RNA is in trace amount) and the stoichiometry (when RNA is not in trace amount) of the protein component required to form complexes following principles described before90. Hill coefficient was calculated as described before89. RNA-EMSA with short (38-mer) oligonucleotide (oligo) probes were performed as described before36,91, with some modifications. Sequences of the oligonucleotides are listed in Supplementary Table3. Briefly, [-32P]ATP labeled RNA oligos were incubated with 101000nM of purified protein in a binding buffer containing 10mM Tris-HCl buffer (pH 7.6), 15mM KCl, 5mM MgCl2, 0.1mM DTT, 10 U of RNase inhibitor, 1g BSA, and 0.2mg/ml yeast tRNA in a 1020l reaction volume. After a 10-min incubation at room temperature RNA-protein complexes were resolved on a 5% non-denaturing polyacrylamide gel at 120V using 0.5x Tris-borate-EDTA as the running buffer at 4C. For titration assays with short oligos the reaction mix was prepared without yeast tRNA. Gels were fixed in an Acetone: Methanol: H2O (10:50:40) solution for 10min, exposed to a Phosphor screen for 1216h and scanned using Typhoon FLA 7000 phosphorimager.

Lung tissues from freshly euthanized uninfected and SARS-CoV-2 infected hamsters were used for DNA damage analysis. Genomic DNA was extracted using the Genomic tip 20/G kit (Qiagen) per the manufacturers protocol, to ensure minimal DNA oxidation during the isolation steps. The DNA was quantitated by Pico Green (Molecular Probes) in a black-bottomed 96-well plate and gene-specific LA-qPCR assays were performed as described earlier33,39 using LongAmp Taq DNA Polymerase (New England Biolabs, Catalog no. M0323). The LA-qPCR reaction was set for all genes from the same stock of diluted genomic DNA sample, to avoid variations in PCR amplification during sample preparation. Preliminary optimization of the assays was performed to ensure the linearity of PCR amplification with respect to the number of cycles and DNA concentration (1015ng). The final PCR reaction conditions were optimized at 94C for 30s; (94C for 30s, 5560C for 30s depending on the oligo annealing temperature, 65C for 10min) for 25 cycles; 65C for 10min. Since amplification of a small region is independent of DNA damage, a small DNA fragment (~200500bp) from the corresponding gene(s) was also amplified for normalization of amplification of the large fragment. Primer sequences used in the assay are listed in Supplementary Table1. The amplified products were then visualized on gels and quantitated with ImageJ software (NIH). The extent of damage was calculated in terms of relative band intensity with the uninfected control mice/hamster sample considered as 100.

All statistical tests were performed using R version 3.2.3 (2015-12-10). Standard t-tests were performed using python scipy.stats.ttest_ind package (version 0.19.0) with Welchs Two Sample t-test (unpaired, unequal variance (equal_var=False), and unequal sample size) parameters. Multiple hypothesis correction was performed by adjusting p values with statsmodels.stats.multitest.multipletests (fdr_bh: Benjamini/Hochberg principles). The results were independently validated with R statistical software (R version 3.6.1; 2019-07-05). Pathway analysis of gene lists were carried out via the Reactome database and algorithm. Reactome identifies signaling and metabolic molecules and organizes their relations into biological pathways and processes. Kaplan-Meier analysis was performed using lifelines python package version 0.14.6. Violin and Swarm plots were created using python seaborn package version 0.10.1.

Graph generation and analysis of statistical significance between two sets of data were performed with Microsoft excel, GraphPad Software (https://www.graphpad.com/quickcalcs/pvalue1.cfm) and MedCalc statistical software (https://www.medcalc.org/calc/comparison_of_means.php). p=values<0.05 were considered statistically significant.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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The DNA glycosylase NEIL2 is protective during SARS-CoV-2 infection - Nature.com

Tracy Morgan and Nas may already be friends, but a new revelation during a DNA test led them to discover an even deeper connection.

During a conversation on the Connect the Dots podcast, Morgan detailed his upcoming appearance on PBS' genealogy showFinding Your Roots, where a DNA test revealed that the rap legend is actually his third cousin.

"I turn the last page, and guess who's sitting there? Nas. Me and Nas is third cousins on my mom's side," Morgan, 55, revealed during the podcast appearance.

"But me and Esco was always tight before that," he added. "I did a show years ago on Comedy Central called One Mic, that was for Nas' mom that just passed away. So me and Esco always been tight."

Morgan who is scheduled to appear on the eighth episode of the PBS series' upcoming tenth season, titled "Mean Streets" revealed that he later called up his longtime pal to share the surprising news.

"When I found out on the West Side Highway, after the show, I called him up and I say, 'Yo Esco,' he said 'What up Trey?' I said, 'Guess what?' He said, 'What?' And I said, 'I just did Finding Your Roots, me and you related,'" Morgan remembered telling Nas, 50.

"He started crying, I started crying. And I said to him, 'If you ever need me, I'm there, cuz.' He said, 'Cuz, if you ever need me I'm there.'"

Stephen Lovekin/FilmMagic

As Morgan noted, the episode is scheduled to air in February, and he learned a few other facts about his familial history in the process.

"They went back 400 years on my father's side and 400 years on my mother's side," he said. "I thought I was big in my life till I found out what my great, great, great grandmother did. My great, great, great, great, great grandfather's name was Abraham Mack. I know the name of the slave masters who owned us I got it right here on my phone and the slave ship."

He continued, "You need to know who you come from before you leave this earth. Know who you are and where you come from. Knowledge itself. I did a lot of crying. And no matter who you are, you're gonna break down."

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Several other celebrities have appeared on Finding Your Roots, including Maya Rudolph, Neil Patrick Harris and Julia Roberts. And Morgan and Nas aren't the only two famous faces to find a DNA connection through the show.

Back in May, Terry CrewsandBilly Crudup met up for the first time as family after discovering they were relatives. Months before that, Bill Hader detailed an email he received from Carol Burnett where she told him that they were also cousins.

The next season of Finding Your Roots with Henry Louis Gates Jr. will run 10 episodes. It premieres Jan. 2 on PBS.

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Tracy Morgan Discovered He Was Related to Nas After DNA Test - PEOPLE

Scientists from a private laboratory who pioneered forensic DNA testing testified Monday about how and why a condom collected as evidence in 1986 near the body of strangulation victim Ruby Doss was discarded three years later after DNA had been extracted.

The handling of DNA evidence has emerged as a key part of suspected killer Richard Aguirres defense.

The testimony of scientists Michael Baird and Lisa Bennett came during the third week of Aguirres trial in Spokane. The two worked at LifeCodes, a private laboratory on the cutting edge of forensic DNA science in the late 1980s, Baird said.

It was really at the beginning of DNA testing for identification in forensic testing, he said.

Baird was involved in developing the protocols for the lab, he said, which included guards against contamination. The lab was not credentialed in forensic DNA testing in 1989 because, he said, that type of credential did not exist yet.

At the time they used restriction fragment length polymorphism testing, Baird said.

That required a microgram of DNA. Now the most common type of DNA testing requires about 1,000 times less DNA to be successful, Baird said.

Bennett conducted the DNA testing on the condom in 1989. She testified she attempted to obtain a DNA print from the condom.

Upon examining the condom, she could not see any semen with the naked eye, so she put the entire condom into a test tube with clarified water to wash off the DNA from the inside and outside of the condom, which she said was not out of the ordinary for the time.

Bennett then disposed of the condom and continued working with the solution that contained the DNA.

I felt I got all the DNA off that I was going to get off by processing it per protocol, Bennett said.

She attempted to separate the sperm DNA and nonsperm DNA, but ultimately, there was not enough DNA for testing with the technology available at the time, Bennett said.

Aguirres attorney, Karen Lindholdt, questioned Bennett extensively on reagent blanks, which are vials of the solutions used in testing kept separately to show a lack of contamination.

Bennett and Baird said it was not common to keep blanks like that at the time, despite being required now.

While Lindholdt asked numerous questions related to contamination of evidence and documentation of chain of custody, Bennett was firm that nothing was amiss in her handling of the evidence based on protocols of the time.

Im not aware of any case that I violated any protocol processing or that I mixed things, Bennett said.

LifeCodes protocols were not available for the prosecution or defense to examine, which Lindholdt argued made it impossible to know if procedure of the time was followed.

Following the scientists testimony, Lindholdt renewed her motion to exclude the DNA evidence, noting the lack of protocol document and lack of accreditation, among other issues.

The fact is, there were lots of chances for contamination of the condom bag, Lindholdt argued. That lab is the genesis of everything we are talking about in this case, and it was 1989; it wasnt accredited.

Prosecutor Larry Haskell argued that it was the inception of DNA testing, and that time has faded the scientists memories and made the protocol unavailable, all of which could go to the weight of the evidence but not exclude it.

He also noted that more testimony regarding contamination was expected.

Spokane County Superior Court Judge Jeremy Schmidt ruled that evidence will remain admitted and he would consider the issues raised by Lindholdt as part of the weight he will give the evidence.

Huma Nasir, who conducted DNA testing on the case in 2008 while working at private lab Orchid Cellmark, also testified.

She found a full male DNA profile in the condom extracts, Nasir said. That profile later matched Aguirre.

Nasir also found partial male DNA profiles on Doss pants and her left hand. Those did not match Aguirre.

Nasir was unable to obtain a profile from the female portion of the condom extracts.

Additional testimony on the DNA evidence is expected Tuesday. Aguirres trial is scheduled to continue through mid-December.

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Scientists testify about DNA in third week of Aguirre trial - The Spokesman Review