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126-400: In genetics , complementary DNA ( cDNA ) is DNA that was reverse transcribed (via reverse transcriptase ) from an RNA (e.g., messenger RNA or microRNA ). cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms. In engineered forms, it often is a copy (replicate) of the naturally occurring DNA from any particular organism's natural genome;
252-436: A complementary DNA (cDNA) library for sequencing are described below, but often vary between platforms. The cDNA library derived from RNA biotypes is then sequenced into a computer-readable format. There are many high-throughput sequencing technologies for cDNA sequencing including platforms developed by Illumina , Thermo Fisher , BGI/MGI , PacBio , and Oxford Nanopore Technologies . For Illumina short-read sequencing,
378-429: A helical structure (i.e., shaped like a corkscrew). Their double-helix model had two strands of DNA with the nucleotides pointing inward, each matching a complementary nucleotide on the other strand to form what look like rungs on a twisted ladder. This structure showed that genetic information exists in the sequence of nucleotides on each strand of DNA. The structure also suggested a simple method for replication : if
504-534: A sexual process for transferring DNA from one cell to another cell (usually of the same species). Transformation requires the action of numerous bacterial gene products , and its primary adaptive function appears to be repair of DNA damages in the recipient cell. The diploid nature of chromosomes allows for genes on different chromosomes to assort independently or be separated from their homologous pair during sexual reproduction wherein haploid gametes are formed. In this way new combinations of genes can occur in
630-573: A DNA molecule. In 1983, Kary Banks Mullis developed the polymerase chain reaction , providing a quick way to isolate and amplify a specific section of DNA from a mixture. The efforts of the Human Genome Project , Department of Energy, NIH, and parallel private efforts by Celera Genomics led to the sequencing of the human genome in 2003. At its most fundamental level, inheritance in organisms occurs by passing discrete heritable units, called genes , from parents to offspring. This property
756-453: A cell and identifying rare transcripts. The reverse transcription step is critical as the efficiency of the RT reaction determines how much of the cell's RNA population will be eventually analyzed by the sequencer. The processivity of reverse transcriptases and the priming strategies used may affect full-length cDNA production and the generation of libraries biased toward the 3’ or 5' end of genes. In
882-474: A common technology for cDNA sequencing, adapters are ligated to the cDNA, DNA is attached to a flow cell, clusters are generated through cycles of bridge amplification and denaturing, and sequence-by-synthesis is performed in cycles of complementary strand synthesis and laser excitation of bases with reversible terminators. Sequencing platform choice and parameters are guided by experimental design and cost. Common experimental design considerations include deciding on
1008-486: A complex trait is called heritability . Measurement of the heritability of a trait is relative—in a more variable environment, the environment has a bigger influence on the total variation of the trait. For example, human height is a trait with complex causes. It has a heritability of 89% in the United States. In Nigeria, however, where people experience a more variable access to good nutrition and health care , height has
1134-483: A different parent. Many species have so-called sex chromosomes that determine the sex of each organism. In humans and many other animals, the Y chromosome contains the gene that triggers the development of the specifically male characteristics. In evolution, this chromosome has lost most of its content and also most of its genes, while the X chromosome is similar to the other chromosomes and contains many genes. This being said, Mary Frances Lyon discovered that there
1260-546: A diploid cell with paired chromosomes. Diploid organisms form haploids by dividing, without replicating their DNA, to create daughter cells that randomly inherit one of each pair of chromosomes. Most animals and many plants are diploid for most of their lifespan, with the haploid form reduced to single cell gametes such as sperm or eggs . Although they do not use the haploid/diploid method of sexual reproduction, bacteria have many methods of acquiring new genetic information. Some bacteria can undergo conjugation , transferring
1386-429: A gene is used to produce a specific amino acid sequence . This process begins with the production of an RNA molecule with a sequence matching the gene's DNA sequence, a process called transcription . This messenger RNA molecule then serves to produce a corresponding amino acid sequence through a process called translation . Each group of three nucleotides in the sequence, called a codon , corresponds either to one of
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#17328764502911512-482: A heritability of only 62%. The molecular basis for genes is deoxyribonucleic acid (DNA). DNA is composed of deoxyribose (sugar molecule), a phosphate group, and a base (amine group). There are four types of bases: adenine (A), cytosine (C), guanine (G), and thymine (T). The phosphates make phosphodiester bonds with the sugars to make long phosphate-sugar backbones. Bases specifically pair together (T&A, C&G) between two backbones and make like rungs on
1638-435: A higher body temperature. In a low-temperature environment, however, the protein's structure is stable and produces dark-hair pigment normally. The protein remains functional in areas of skin that are colder—such as its legs, ears, tail, and face—so the cat has dark hair at its extremities. Environment plays a major role in effects of the human genetic disease phenylketonuria . The mutation that causes phenylketonuria disrupts
1764-503: A ladder. The bases, phosphates, and sugars together make a nucleotide that connects to make long chains of DNA. Genetic information exists in the sequence of these nucleotides, and genes exist as stretches of sequence along the DNA chain. These chains coil into a double a-helix structure and wrap around proteins called Histones which provide the structural support. DNA wrapped around these histones are called chromosomes. Viruses sometimes use
1890-505: A living cell or organism may increase or decrease gene transcription. A classic example is two seeds of genetically identical corn, one placed in a temperate climate and one in an arid climate (lacking sufficient waterfall or rain). While the average height the two corn stalks could grow to is genetically determined, the one in the arid climate only grows to half the height of the one in the temperate climate due to lack of water and nutrients in its environment. The word genetics stems from
2016-407: A mistaken alignment; this makes some regions in genomes more prone to mutating in this way. These errors create large structural changes in DNA sequence— duplications , inversions , deletions of entire regions—or the accidental exchange of whole parts of sequences between different chromosomes, chromosomal translocation . RNA-Seq RNA-Seq (named as an abbreviation of RNA sequencing )
2142-456: A phenotype involves studying identical and fraternal twins , or other siblings of multiple births . Identical siblings are genetically the same since they come from the same zygote. Meanwhile, fraternal twins are as genetically different from one another as normal siblings. By comparing how often a certain disorder occurs in a pair of identical twins to how often it occurs in a pair of fraternal twins, scientists can determine whether that disorder
2268-544: A possible fusion event, however, because of the length of the reads, this could prove to be very noisy. An alternative approach is to use paired-end reads, when a potentially large number of paired reads would map each end to a different exon, giving better coverage of these events (see figure). Nonetheless, the end result consists of multiple and potentially novel combinations of genes providing an ideal starting point for further validation. Copy number alteration (CNA) analyses are commonly used in cancer studies. Gain and loss of
2394-509: A reverse transcriptase enzyme and purified RNA templates, one strand of cDNA is produced (first-strand cDNA synthesis). The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reduced RNase H activity suited for transcription of longer RNAs. The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature). cDNA
2520-403: A series of genes can be combined to form a linear linkage map that roughly describes the arrangement of the genes along the chromosome. Genes express their functional effect through the production of proteins, which are molecules responsible for most functions in the cell. Proteins are made up of one or more polypeptide chains, each composed of a sequence of amino acids . The DNA sequence of
2646-421: A similar way across tissues and experimental conditions. Their main purpose lies in hypothesis generation and guilt-by-association approaches for inferring functions of previously unknown genes. RNA-Seq data has been used to infer genes involved in specific pathways based on Pearson correlation , both in plants and mammals. The main advantage of RNA-Seq data in this kind of analysis over the microarray platforms
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#17328764502912772-457: A single parent. Offspring that are genetically identical to their parents are called clones . Eukaryotic organisms often use sexual reproduction to generate offspring that contain a mixture of genetic material inherited from two different parents. The process of sexual reproduction alternates between forms that contain single copies of the genome ( haploid ) and double copies ( diploid ). Haploid cells fuse and combine genetic material to create
2898-409: A size exclusion gel, through size selection magnetic beads, or with a commercially developed kit. Once isolated, linkers are added to the 3' and 5' end then purified. The final step is cDNA generation through reverse transcription. Because converting RNA into cDNA , ligation, amplification, and other sample manipulations have been shown to introduce biases and artifacts that may interfere with both
3024-448: A small circular piece of DNA to another bacterium. Bacteria can also take up raw DNA fragments found in the environment and integrate them into their genomes, a phenomenon known as transformation . These processes result in horizontal gene transfer , transmitting fragments of genetic information between organisms that would be otherwise unrelated. Natural bacterial transformation occurs in many bacterial species, and can be regarded as
3150-400: A smooth blend of traits from their parents. Mendel's work provided examples where traits were definitely not blended after hybridization, showing that traits are produced by combinations of distinct genes rather than a continuous blend. Blending of traits in the progeny is now explained by the action of multiple genes with quantitative effects . Another theory that had some support at that time
3276-456: A specific protein can be transferred to a recipient cell for expression as part of recombinant DNA , often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays , qPCR , and RNA-seq . In natural forms, cDNA is produced by retroviruses (such as HIV-1 , HIV-2 , simian immunodeficiency virus , etc.) and then integrated into
3402-583: A strong promoter to drive transcription of the target cDNA into mRNA, which is then translated into protein. cDNA is also used to study gene expression via methods such as RNA-seq or RT-qPCR . For sequencing, RNA must be fragmented due to sequencing platform size limitations. Additionally, second-strand synthesized cDNA must be ligated with adapters that allow cDNA fragments to be PCR amplified and bind to sequencing flow cells. Gene-specific analysis methods commonly use microarrays and RT-qPCR to quantify cDNA levels via fluorometric and other methods. On 13 June 2013,
3528-448: A transition of heredity from its status as myth to that of a scientific discipline, by providing a fundamental theoretical basis for genetics in the twentieth century. Other theories of inheritance preceded Mendel's work. A popular theory during the 19th century, and implied by Charles Darwin 's 1859 On the Origin of Species , was blending inheritance : the idea that individuals inherit
3654-412: A unique three-dimensional structure for that protein, and the three-dimensional structures of proteins are related to their functions. Some are simple structural molecules, like the fibers formed by the protein collagen . Proteins can bind to other proteins and simple molecules, sometimes acting as enzymes by facilitating chemical reactions within the bound molecules (without changing the structure of
3780-400: A variety of hereditary characteristics that replicate and remain active throughout generations. While haploid organisms have only one copy of each chromosome, most animals and many plants are diploid , containing two of each chromosome and thus two copies of every gene. The two alleles for a gene are located on identical loci of the two homologous chromosomes , each allele inherited from
3906-488: Is X-chromosome inactivation during reproduction to avoid passing on twice as many genes to the offspring. Lyon's discovery led to the discovery of X-linked diseases. When cells divide, their full genome is copied and each daughter cell inherits one copy. This process, called mitosis , is the simplest form of reproduction and is the basis for asexual reproduction. Asexual reproduction can also occur in multicellular organisms, producing offspring that inherit their genome from
Complementary DNA - Misplaced Pages Continue
4032-1137: Is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome . Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts , post-transcriptional modifications , gene fusion , mutations/ SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA , tRNA , and ribosomal profiling . RNA-Seq can also be used to determine exon / intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing , bulk RNA sequencing , 3' mRNA-sequencing , in situ sequencing of fixed tissue, and native RNA molecule sequencing with single-molecule real-time sequencing. Other examples of emerging RNA-Seq applications due to
4158-435: Is a very rare event. Once reverse transcription is complete, the cDNAs from many cells can be mixed together for sequencing; transcripts from a particular cell are identified by each cell's unique barcode. Unique molecular identifier (UMIs) can be attached to mRNA/cDNA target sequences to help identify artifacts during library preparation. Challenges for scRNA-Seq include preserving the initial relative abundance of mRNA in
4284-422: Is a weighted sum of expression of all genes in a module. Eigengenes are useful biomarkers (features) for diagnosis and prognosis. Variance-Stabilizing Transformation approaches for estimating correlation coefficients based on RNA seq data have been proposed. RNA-Seq captures DNA variation, including single nucleotide variants , small insertions/deletions . and structural variation . Variant calling in RNA-Seq
4410-417: Is achieved by designing sequence-specific DNA primers that hybridize to the 5' and 3' ends of a cDNA region coding for a protein. Once amplified, the sequence can be cut at each end with nucleases and inserted into one of many small circular DNA sequences known as expression vectors. Such vectors allow for self-replication, inside the cells, and potentially integration in the host DNA. They typically also contain
4536-478: Is caused by genetic or postnatal environmental factors. One famous example involved the study of the Genain quadruplets , who were identical quadruplets all diagnosed with schizophrenia . The genome of a given organism contains thousands of genes, but not all these genes need to be active at any given moment. A gene is expressed when it is being transcribed into mRNA and there exist many cellular methods of controlling
4662-476: Is commonly generated from mRNA for gene expression analyses such as RT-qPCR and RNA-seq . mRNA is selectively reverse transcribed using oligo-d T primers that are the reverse complement of the poly-adenylated tail on the 3' end of all mRNA. The oligo-dT primer anneals to the poly-adenylated tail of the mRNA to serve as a binding site for the reverse transcriptase to begin reverse transcription. An optimized mixture of oligo-dT and random hexamer primers increases
4788-437: Is finding differences in gene expression between two or more conditions ( e.g. , treated vs not treated); this process is called differential expression. The outputs are frequently referred to as differentially expressed genes (DEGs) and these genes can either be up- or down-regulated ( i.e. , higher or lower in the condition of interest). There are many tools that perform differential expression . Most are run in R , Python , or
4914-711: Is in part dictated by costs in money and time; a related limitation is the required team of specialists (bioinformaticians, physicians/clinicians, basic researchers, technicians) to fully interpret the huge amount of data generated by this analysis. A lot of emphasis has been given to RNA-Seq data after the Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA) projects have used this approach to characterize dozens of cell lines and thousands of primary tumor samples, respectively. ENCODE aimed to identify genome-wide regulatory regions in different cohort of cell lines and transcriptomic data are paramount to understand
5040-485: Is maintained by inactivating RNases with chaotropic agents such as guanidinium isothiocyanate, sodium dodecyl sulphate (SDS), phenol or chloroform. Total RNA is then separated from other cellular components and precipitated with alcohol. Various commercial kits exist for simple and rapid RNA extractions for specific applications. Additional bead-based methods can be used to isolate specific sub-types of RNA (e.g. mRNA and microRNA ) based on size or unique RNA regions. Using
5166-407: Is patent-eligible, whereas isolated sequences of naturally occurring DNA comprising introns are not. RNA serves as a template for cDNA synthesis. In cellular life, cDNA is generated by viruses and retrotransposons for integration of RNA into target genomic DNA . In molecular biology, RNA is purified from source material after genomic DNA, proteins and other cellular components are removed. cDNA
Complementary DNA - Misplaced Pages Continue
5292-1009: Is quantified by counting the number of reads that mapped to each locus in the transcriptome assembly step. Expression can be quantified for exons or genes using contigs or reference transcript annotations. These observed RNA-Seq read counts have been robustly validated against older technologies, including expression microarrays and qPCR . Tools that quantify counts are HTSeq, FeatureCounts, Rcount, maxcounts, FIXSEQ, and Cuffquant. These tools determine read counts from aligned RNA-Seq data, but alignment-free counts can also be obtained with Sailfish and Kallisto. The read counts are then converted into appropriate metrics for hypothesis testing, regressions, and other analyses. Parameters for this conversion are: RNA spike-ins are samples of RNA at known concentrations that can be used as gold standards in experimental design and during downstream analyses for absolute quantification and detection of genome-wide effects. The simplest but often most powerful use of RNA-Seq
5418-441: Is responsible for the development of structures within multicellular organisms, these patterns arise from the complex interactions between many cells. Within eukaryotes , there exist structural features of chromatin that influence the transcription of genes, often in the form of modifications to DNA and chromatin that are stably inherited by daughter cells. These features are called " epigenetic " because they exist "on top" of
5544-498: Is shared with viruses with the exclusion of the generation of infectious particles. Mark D. Adams et al. "Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project." Science (American Association for the Advancement of Science) 252.5013 (1991): 1651–1656. Web. Philip M. Murphy, and H. Lee Tiffany. "Cloning of Complementary DNA Encoding a Functional Human Interleukin-8 Receptor." Science (American Association for
5670-548: Is similar to DNA variant calling and often employs the same tools (including SAMtools mpileup and GATK HaplotypeCaller ) with adjustments to account for splicing. One unique dimension for RNA variants is allele-specific expression (ASE) : the variants from only one haplotype might be preferentially expressed due to regulatory effects including imprinting and expression quantitative trait loci , and noncoding rare variants . Limitations of RNA variant identification include that it only reflects expressed regions (in humans, <5% of
5796-638: Is that transcripts can be covered in full length, allowing for higher confidence isoform detection and quantification compared to short-read sequencing. Traditionally, single-molecule RNA-Seq methods have higher error rates compared to short-read sequencing, but newer methods like ONT direct RNA-Seq limit errors by avoiding fragmentation and cDNA conversion. Recent uses of ONT direct RNA-Seq for differential expression in human cell populations have demonstrated that this technology can overcome many limitations of short and long cDNA sequencing. Standard methods such as microarrays and standard bulk RNA-Seq analysis analyze
5922-628: Is the capability to cover the entire transcriptome, therefore allowing the possibility to unravel more complete representations of the gene regulatory networks. Differential regulation of the splice isoforms of the same gene can be detected and used to predict their biological functions. Weighted gene co-expression network analysis has been successfully used to identify co-expression modules and intramodular hub genes based on RNA seq data. Co-expression modules may correspond to cell types or pathways. Highly connected intramodular hubs can be interpreted as representatives of their respective module. An eigengene
6048-497: Is the physical basis for inheritance: DNA replication duplicates the genetic information by splitting the strands and using each strand as a template for synthesis of a new partner strand. Genes are arranged linearly along long chains of DNA base-pair sequences. In bacteria , each cell usually contains a single circular genophore , while eukaryotic organisms (such as plants and animals) have their DNA arranged in multiple linear chromosomes. These DNA strands are often extremely long;
6174-694: Is the same as that which Mendel published. In his third law, he developed the basic principles of mutation (he can be considered a forerunner of Hugo de Vries ). Festetics argued that changes observed in the generation of farm animals, plants, and humans are the result of scientific laws. Festetics empirically deduced that organisms inherit their characteristics, not acquire them. He recognized recessive traits and inherent variation by postulating that traits of past generations could reappear later, and organisms could produce progeny with different attributes. These observations represent an important prelude to Mendel's theory of particulate inheritance insofar as it features
6300-686: Is then synthesized through in vitro reverse transcription . RNA is transcribed from genomic DNA in host cells and is extracted by first lysing cells then purifying RNA utilizing widely used methods such as phenol-chloroform, silica column, and bead-based RNA extraction methods. Extraction methods vary depending on the source material. For example, extracting RNA from plant tissue requires additional reagents, such as polyvinylpyrrolidone (PVP), to remove phenolic compounds, carbohydrates, and other compounds that will otherwise render RNA unusable. To remove DNA and proteins, enzymes such as DNase and Proteinase K are used for degradation. Importantly, RNA integrity
6426-634: Is to first look for enrichment of known biology as a sanity check and then expand the scope to look for novel biology. RNA splicing is integral to eukaryotes and contributes significantly to protein regulation and diversity, occurring in >90% of human genes. There are multiple alternative splicing modes : exon skipping (most common splicing mode in humans and higher eukaryotes), mutually exclusive exons, alternative donor or acceptor sites, intron retention (most common splicing mode in plants, fungi, and protozoa), alternative transcription start site (promoter), and alternative polyadenylation. One goal of RNA-Seq
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#17328764502916552-602: Is to identify alternative splicing events and test if they differ between conditions. Long-read sequencing captures the full transcript and thus minimizes many of issues in estimating isoform abundance, like ambiguous read mapping. For short-read RNA-Seq, there are multiple methods to detect alternative splicing that can be classified into three main groups: Differential gene expression tools can also be used for differential isoform expression if isoforms are quantified ahead of time with other tools like RSEM. Coexpression networks are data-derived representations of genes behaving in
6678-479: The Blue-eyed Mary ( Omphalodes verna ), for example, there exists a gene with alleles that determine the color of flowers: blue or magenta. Another gene, however, controls whether the flowers have color at all or are white. When a plant has two copies of this white allele, its flowers are white—regardless of whether the first gene has blue or magenta alleles. This interaction between genes is called epistasis , with
6804-530: The United States Supreme Court ruled in the case of Association for Molecular Pathology v. Myriad Genetics that while naturally occurring genes cannot be patented , cDNA is patent-eligible because it does not occur naturally. Some viruses also use cDNA to turn their viral RNA into mRNA (viral RNA → cDNA → mRNA). The mRNA is used to make viral proteins to take over the host cell. An example of this first step from viral RNA to cDNA can be seen in
6930-543: The Unix command line. Commonly used tools include DESeq, edgeR, and voom+limma, all of which are available through R/ Bioconductor . These are the common considerations when performing differential expression: Downstream analyses for a list of differentially expressed genes come in two flavors, validating observations and making biological inferences. Owing to the pitfalls of differential expression and RNA-Seq, important observations are replicated with (1) an orthogonal method in
7056-413: The ancient Greek γενετικός genetikos meaning "genitive"/"generative", which in turn derives from γένεσις genesis meaning "origin". The observation that living things inherit traits from their parents has been used since prehistoric times to improve crop plants and animals through selective breeding . The modern science of genetics, seeking to understand this process, began with
7182-415: The neutral theory of molecular evolution through publishing the nearly neutral theory of molecular evolution . In this theory, Ohta stressed the importance of natural selection and the environment to the rate at which genetic evolution occurs. One important development was chain-termination DNA sequencing in 1977 by Frederick Sanger . This technology allows scientists to read the nucleotide sequence of
7308-497: The 2018 Breakthrough of the Year . A variety of parameters are considered when designing and conducting RNA-Seq experiments: Two methods are used to assign raw sequence reads to genomic features (i.e., assemble the transcriptome): A note on assembly quality: The current consensus is that 1) assembly quality can vary depending on which metric is used, 2) assembly tools that scored well in one species do not necessarily perform well in
7434-477: The 3' end of the first-strand cDNA to prime second-strand synthesis. However, priming is random and hairpin hydrolysis leads to loss of information. The Gubler and Hoffman Procedure uses E. Coli RNase H to nick mRNA that is replaced with E. Coli DNA Polymerase I and sealed with E. Coli DNA Ligase . An optimization of this procedure relies on low RNase H activity of M-MLV to nick mRNA with remaining RNA later removed by adding RNase H after DNA Polymerase translation of
7560-503: The Advancement of Science) 253.5025 (1991): 1280–1283. Web. Genetics Genetics is the study of genes , genetic variation , and heredity in organisms . It is an important branch in biology because heredity is vital to organisms' evolution . Gregor Mendel , a Moravian Augustinian friar working in the 19th century in Brno , was the first to study genetics scientifically. Mendel studied "trait inheritance", patterns in
7686-421: The DNA sequence and retain inheritance from one cell generation to the next. Because of epigenetic features, different cell types grown within the same medium can retain very different properties. Although epigenetic features are generally dynamic over the course of development, some, like the phenomenon of paramutation , have multigenerational inheritance and exist as rare exceptions to the general rule of DNA as
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#17328764502917812-624: The F1 offspring mate with each other, the offspring are called the "F2" (second filial) generation. One of the common diagrams used to predict the result of cross-breeding is the Punnett square . When studying human genetic diseases, geneticists often use pedigree charts to represent the inheritance of traits. These charts map the inheritance of a trait in a family tree. Organisms have thousands of genes, and in sexually reproducing organisms these genes generally assort independently of each other. This means that
7938-496: The Greek word genesis —γένεσις, "origin", predates the noun and was first used in a biological sense in 1860. Bateson both acted as a mentor and was aided significantly by the work of other scientists from Newnham College at Cambridge, specifically the work of Becky Saunders , Nora Darwin Barlow , and Muriel Wheldale Onslow . Bateson popularized the usage of the word genetics to describe
8064-618: The HIV cycle of infection. Here, the host cell membrane becomes attached to the virus' lipid envelope which allows the viral capsid with two copies of viral genome RNA to enter the host. The cDNA copy is then made through reverse transcription of the viral RNA, a process facilitated by the chaperone CypA and a viral capsid associated reverse transcriptase. cDNA is also generated by retrotransposons in eukaryotic genomes. Retrotransposons are mobile genetic elements that move themselves within, and sometimes between, genomes via RNA intermediates. This mechanism
8190-448: The ability of the body to break down the amino acid phenylalanine , causing a toxic build-up of an intermediate molecule that, in turn, causes severe symptoms of progressive intellectual disability and seizures. However, if someone with the phenylketonuria mutation follows a strict diet that avoids this amino acid, they remain normal and healthy. A common method for determining how genes and environment ("nature and nurture") contribute to
8316-444: The adaptive function of repair of DNA damages. The first cytological demonstration of crossing over was performed by Harriet Creighton and Barbara McClintock in 1931. Their research and experiments on corn provided cytological evidence for the genetic theory that linked genes on paired chromosomes do in fact exchange places from one homolog to the other. The probability of chromosomal crossover occurring between two given points on
8442-414: The advancement of bioinformatics algorithms are copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays . Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know
8568-829: The amplification step, either PCR or in vitro transcription (IVT) is currently used to amplify cDNA. One of the advantages of PCR-based methods is the ability to generate full-length cDNA. However, different PCR efficiency on particular sequences (for instance, GC content and snapback structure) may also be exponentially amplified, producing libraries with uneven coverage. On the other hand, while libraries generated by IVT can avoid PCR-induced sequence bias, specific sequences may be transcribed inefficiently, thus causing sequence drop-out or generating incomplete sequences. Several scRNA-Seq protocols have been published: Tang et al., STRT, SMART-seq, CEL-seq, RAGE-seq, Quartz-seq and C1-CAGE. These protocols differ in terms of strategies for reverse transcription, cDNA synthesis and amplification, and
8694-440: The basis for inheritance. During the process of DNA replication, errors occasionally occur in the polymerization of the second strand. These errors, called mutations, can affect the phenotype of an organism, especially if they occur within the protein coding sequence of a gene. Error rates are usually very low—1 error in every 10–100 million bases—due to the "proofreading" ability of DNA polymerases . Processes that increase
8820-404: The cat plays the role of the environment. The cat's genes code for dark hair, thus the hair-producing cells in the cat make cellular proteins resulting in dark hair. But these dark hair-producing proteins are sensitive to temperature (i.e. have a mutation causing temperature-sensitivity) and denature in higher-temperature environments, failing to produce dark-hair pigment in areas where the cat has
8946-429: The cell, these genes for tryptophan synthesis are no longer needed. The presence of tryptophan directly affects the activity of the genes—tryptophan molecules bind to the tryptophan repressor (a transcription factor), changing the repressor's structure such that the repressor binds to the genes. The tryptophan repressor blocks the transcription and expression of the genes, thereby creating negative feedback regulation of
9072-442: The chance of obtaining full-length cDNA while reducing 5' or 3' bias. Ribosomal RNA may also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as some non-coding RNA . The result of first-strand syntheses, RNA-DNA hybrids, can be processed through multiple second-strand synthesis methods or processed directly in downstream assays. An early method known as hairpin-primed synthesis relied on hairpin formation on
9198-421: The chromosome is related to the distance between the points. For an arbitrarily long distance, the probability of crossover is high enough that the inheritance of the genes is effectively uncorrelated. For genes that are closer together, however, the lower probability of crossover means that the genes demonstrate genetic linkage; alleles for the two genes tend to be inherited together. The amounts of linkage between
9324-422: The combined DNA sequences of all chromosomes) is called the genome . DNA is most often found in the nucleus of cells, but Ruth Sager helped in the discovery of nonchromosomal genes found outside of the nucleus. In plants, these are often found in the chloroplasts and in other organisms, in the mitochondria. These nonchromosomal genes can still be passed on by either partner in sexual reproduction and they control
9450-426: The development of embryos and organisms, including the worm Caenorhabditis elegans , and the regenerative planarian Schmidtea mediterranea . The first vertebrate animals to be mapped in this way were Zebrafish and Xenopus laevis . In each case multiple stages of the embryo were studied, allowing the entire process of development to be mapped on a cell-by-cell basis. Science recognized these advances as
9576-423: The downstream effect of those epigenetic and genetic regulatory layers. TCGA, instead, aimed to collect and analyze thousands of patient's samples from 30 different tumor types to understand the underlying mechanisms of malignant transformation and progression. In this context RNA-Seq data provide a unique snapshot of the transcriptomic status of the disease and look at an unbiased population of transcripts that allows
9702-502: The entire gene), because the DNA for an entire gene may include DNA that does not code for the protein or that interrupts the coding sequence of the protein (e.g., introns ). Partial sequences of cDNAs are often obtained as expressed sequence tags . With amplification of DNA sequences via polymerase chain reaction (PCR) now commonplace, one will typically conduct reverse transcription as an initial step, followed by PCR to obtain an exact sequence of cDNA for intra-cellular expression. This
9828-1004: The existence of rare cell types within a cell population that may never have been seen before. For example, rare specialized cells in the lung called pulmonary ionocytes that express the Cystic fibrosis transmembrane conductance regulator were identified in 2018 by two groups performing scRNA-Seq on lung airway epithelia. Current scRNA-Seq protocols involve the following steps: isolation of single cell and RNA, reverse transcription (RT), amplification, library generation and sequencing. Single cells are either mechanically separated into microwells (e.g., BD Rhapsody, Takara ICELL8, Vycap Puncher Platform, or CellMicrosystems CellRaft) or encapsulated in droplets (e.g., 10x Genomics Chromium, Illumina Bio-Rad ddSEQ, 1CellBio InDrop, Dolomite Bio Nadia). Single cells are labeled by adding beads with barcoded oligonucleotides; both cells and beads are supplied in limited amounts such that co-occupancy with multiple cells and beads
9954-526: The expression of RNAs from large populations of cells. In mixed cell populations, these measurements may obscure critical differences between individual cells within these populations. Single-cell RNA sequencing (scRNA-Seq) provides the expression profiles of individual cells. Although it is not possible to obtain complete information on every RNA expressed by each cell, due to the small amount of material available, patterns of gene expression can be identified through gene clustering analyses . This can uncover
10080-405: The expression of TEs and Neoantigen prediction etc. RNA-Seq was first developed in mid 2000s with the advent of next-generation sequencing technology. The first manuscripts that used RNA-Seq even without using the term includes those of prostate cancer cell lines (dated 2006), Medicago truncatula (2006), maize (2007), and Arabidopsis thaliana (2007), while the term "RNA-Seq" itself
10206-417: The expression of genes such that proteins are produced only when needed by the cell. Transcription factors are regulatory proteins that bind to DNA, either promoting or inhibiting the transcription of a gene. Within the genome of Escherichia coli bacteria, for example, there exists a series of genes necessary for the synthesis of the amino acid tryptophan . However, when tryptophan is already available to
10332-669: The function and behavior of genes. Gene structure and function, variation, and distribution are studied within the context of the cell , the organism (e.g. dominance ), and within the context of a population. Genetics has given rise to a number of subfields, including molecular genetics , epigenetics , and population genetics . Organisms studied within the broad field span the domains of life ( archaea , bacteria , and eukarya ). Genetic processes work in combination with an organism's environment and experiences to influence development and behavior , often referred to as nature versus nurture . The intracellular or extracellular environment of
10458-407: The gene's transcript has an allele/variant not observed in the genomic data. Caused by different structural modifications in the genome, fusion genes have gained attention because of their relationship with cancer. The ability of RNA-Seq to analyze a sample's whole transcriptome in an unbiased fashion makes it an attractive tool to find these kinds of common events in cancer. The idea follows from
10584-751: The genes have signalling pathway implications and are a key biomarker of molecular dysfunction in oncology. Calling the CNA information from RNA-Seq data is not straightforward because of the differences in gene expression, which lead to the read depth variance of different magnitudes across genes. Due to these difficulties, most of these analyses are usually done using whole-genome sequencing / whole-exome sequencing (WGS/WES). But advanced bioinformatics tools can call CNA from RNA-Seq. The applications of RNA-Seq are growing day by day. Other new application of RNA-Seq includes detection of microbial contaminants, determining cell type abundance (cell type deconvolution), measuring
10710-399: The genome), could be subject to biases introduced by data processing (e.g., de novo transcriptome assemblies underestimate heterozygosity ), and has lower quality when compared to direct DNA sequencing. Having the matching genomic and transcriptomic sequences of an individual can help detect post-transcriptional edits ( RNA editing ). A post-transcriptional modification event is identified if
10836-534: The host's genome, where it creates a provirus . The term cDNA is also used, typically in a bioinformatics context, to refer to an mRNA transcript's sequence, expressed as DNA bases (deoxy-GCAT) rather than RNA bases (GCAU). Patentability of cDNA was a subject of a 2013 US Supreme Court decision in Association for Molecular Pathology v. Myriad Genetics, Inc. As a compromise, the Court declared, that exons -only cDNA
10962-464: The information an organism uses to function, the environment plays an important role in determining the ultimate phenotypes an organism displays. The phrase " nature and nurture " refers to this complementary relationship. The phenotype of an organism depends on the interaction of genes and the environment. An interesting example is the coat coloration of the Siamese cat . In this case, the body temperature of
11088-406: The inheritance of an allele for yellow or green pea color is unrelated to the inheritance of alleles for white or purple flowers. This phenomenon, known as " Mendel's second law " or the "law of independent assortment," means that the alleles of different genes get shuffled between parents to form offspring with many different combinations. Different genes often interact to influence the same trait. In
11214-427: The largest human chromosome, for example, is about 247 million base pairs in length. The DNA of a chromosome is associated with structural proteins that organize, compact, and control access to the DNA, forming a material called chromatin ; in eukaryotes, chromatin is usually composed of nucleosomes , segments of DNA wound around cores of histone proteins. The full set of hereditary material in an organism (usually
11340-557: The nature of inheritance in plants. In his paper " Versuche über Pflanzenhybriden " (" Experiments on Plant Hybridization "), presented in 1865 to the Naturforschender Verein (Society for Research in Nature) in Brno , Mendel traced the inheritance patterns of certain traits in pea plants and described them mathematically. Although this pattern of inheritance could only be observed for a few traits, Mendel's work suggested that heredity
11466-500: The offspring of a mating pair. Genes on the same chromosome would theoretically never recombine. However, they do, via the cellular process of chromosomal crossover . During crossover, chromosomes exchange stretches of DNA, effectively shuffling the gene alleles between the chromosomes. This process of chromosomal crossover generally occurs during meiosis , a series of cell divisions that creates haploid cells. Meiotic recombination , particularly in microbial eukaryotes , appears to serve
11592-412: The organism's own mRNA was naturally transcribed from its DNA, and the cDNA is reverse transcribed from the mRNA, yielding a duplicate of the original DNA. Engineered cDNA is often used to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), or to sequence or quantify mRNA molecules using DNA based methods (qPCR, RNA-seq). cDNA that codes for
11718-435: The original sequence. A particularly important source of DNA damages appears to be reactive oxygen species produced by cellular aerobic respiration , and these can lead to mutations. In organisms that use chromosomal crossover to exchange DNA and recombine genes, errors in alignment during meiosis can also cause mutations. Errors in crossover are especially likely when similar sequences cause partner chromosomes to adopt
11844-463: The other species, and 3) combining different approaches might be the most reliable. Expression is quantified to study cellular changes in response to external stimuli, differences between healthy and diseased states, and other research questions. Transcript levels are often used as a proxy for protein abundance, but these are often not equivalent due to post transcriptional events such as RNA interference and nonsense-mediated decay . Expression
11970-488: The overlap between two gene sets is statistically significant, in this case the overlap between differentially expressed genes and gene sets from known pathways/databases ( e.g. , Gene Ontology , KEGG , Human Phenotype Ontology ) or from complementary analyses in the same data (like co-expression networks). Common tools for gene set enrichment include web interfaces ( e.g. , ENRICHR, g:profiler, WEBGESTALT) and software packages. When evaluating enrichment results, one heuristic
12096-470: The phenotype of the organism, while the other allele is called recessive as its qualities recede and are not observed. Some alleles do not have complete dominance and instead have incomplete dominance by expressing an intermediate phenotype, or codominance by expressing both alleles at once. When a pair of organisms reproduce sexually , their offspring randomly inherit one of the two alleles from each parent. These observations of discrete inheritance and
12222-519: The possibility to accommodate sequence-specific barcodes (i.e. UMIs ) or the ability to process pooled samples. In 2017, two approaches were introduced to simultaneously measure single-cell mRNA and protein expression through oligonucleotide-labeled antibodies known as REAP-seq, and CITE-seq. scRNA-Seq is becoming widely used across biological disciplines including Development, Neurology , Oncology , Autoimmune disease , and Infectious disease . scRNA-Seq has provided considerable insight into
12348-543: The process of protein production . It was discovered that the cell uses DNA as a template to create matching messenger RNA , molecules with nucleotides very similar to DNA. The nucleotide sequence of a messenger RNA is used to create an amino acid sequence in protein; this translation between nucleotide sequences and amino acid sequences is known as the genetic code . With the newfound molecular understanding of inheritance came an explosion of research. A notable theory arose from Tomoko Ohta in 1973 with her amendment to
12474-415: The process of aligning the short transcriptomic reads to a reference genome. Most of the short reads will fall within one complete exon, and a smaller but still large set would be expected to map to known exon-exon junctions. The remaining unmapped short reads would then be further analyzed to determine whether they match an exon-exon junction where the exons come from different genes. This would be evidence of
12600-435: The product rule, the sum rule, and more. Geneticists use diagrams and symbols to describe inheritance. A gene is represented by one or a few letters. Often a "+" symbol is used to mark the usual, non-mutant allele for a gene. In fertilization and breeding experiments (and especially when discussing Mendel's laws) the parents are referred to as the "P" generation and the offspring as the "F1" (first filial) generation. When
12726-897: The proper characterization and quantification of transcripts, single molecule direct RNA sequencing has been explored by companies including Helicos (bankrupt), Oxford Nanopore Technologies , and others. This technology sequences RNA molecules directly in a massively-parallel manner. Massively parallel single molecule direct RNA-Seq has been explored as an alternative to traditional RNA-Seq, in which RNA-to- cDNA conversion, ligation, amplification, and other sample manipulation steps may introduce biases and artifacts. Technology platforms that perform single-molecule real-time RNA-Seq include Oxford Nanopore Technologies (ONT) Nanopore sequencing , PacBio IsoSeq, and Helicos (bankrupt). Sequencing RNA in its native form preserves modifications like methylation, allowing them to be investigated directly and simultaneously. Another benefit of single-molecule RNA-Seq
12852-519: The properties of a protein by destabilizing the structure or changing the surface of the protein in a way that changes its interaction with other proteins and molecules. For example, sickle-cell anemia is a human genetic disease that results from a single base difference within the coding region for the β-globin section of hemoglobin, causing a single amino acid change that changes hemoglobin's physical properties. Sickle-cell versions of hemoglobin stick to themselves, stacking to form fibers that distort
12978-426: The protein itself). Protein structure is dynamic; the protein hemoglobin bends into slightly different forms as it facilitates the capture, transport, and release of oxygen molecules within mammalian blood. A single nucleotide difference within DNA can cause a change in the amino acid sequence of a protein. Because protein structures are the result of their amino acid sequences, some changes can dramatically change
13104-406: The rate of changes in DNA are called mutagenic : mutagenic chemicals promote errors in DNA replication, often by interfering with the structure of base-pairing, while UV radiation induces mutations by causing damage to the DNA structure. Chemical damage to DNA occurs naturally as well and cells use DNA repair mechanisms to repair mismatches and breaks. The repair does not, however, always restore
13230-408: The same allele of a given gene are called homozygous at that gene locus , while organisms with two different alleles of a given gene are called heterozygous . The set of alleles for a given organism is called its genotype , while the observable traits of the organism are called its phenotype . When organisms are heterozygous at a gene, often one allele is called dominant as its qualities dominate
13356-451: The same samples (like real-time PCR ) or (2) another, sometimes pre-registered , experiment in a new cohort. The latter helps ensure generalizability and can typically be followed up with a meta-analysis of all the pooled cohorts. The most common method for obtaining higher-level biological understanding of the results is gene set enrichment analysis , although sometimes candidate gene approaches are employed. Gene set enrichment determines if
13482-401: The second gene epistatic to the first. Many traits are not discrete features (e.g. purple or white flowers) but are instead continuous features (e.g. human height and skin color ). These complex traits are products of many genes. The influence of these genes is mediated, to varying degrees, by the environment an organism has experienced. The degree to which an organism's genes contribute to
13608-400: The second-strand cDNA. This prevents lost sequence information at the 5' end of the mRNA. Complementary DNA is often used in gene cloning or as gene probes or in the creation of a cDNA library . When scientists transfer a gene from one cell into another cell in order to express the new genetic material as a protein in the recipient cell, the cDNA will be added to the recipient (rather than
13734-506: The segregation of alleles are collectively known as Mendel's first law or the Law of Segregation. However, the probability of getting one gene over the other can change due to dominant, recessive, homozygous, or heterozygous genes. For example, Mendel found that if you cross heterozygous organisms your odds of getting the dominant trait is 3:1. Real geneticist study and calculate probabilities by using theoretical probabilities, empirical probabilities,
13860-419: The sequence a priori . Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed sequence tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression ), and finally to the current technology, next-gen sequencing of complementary DNA (cDNA), notably RNA-Seq. The general steps to prepare
13986-449: The sequencing length, sequencing depth, use of single versus paired-end sequencing, number of replicates, multiplexing, randomization, and spike-ins. When sequencing RNA other than mRNA, the library preparation is modified. The cellular RNA is selected based on the desired size range. For small RNA targets, such as miRNA , the RNA is isolated through size selection. This can be performed with
14112-653: The shape of red blood cells carrying the protein. These sickle-shaped cells no longer flow smoothly through blood vessels , having a tendency to clog or degrade, causing the medical problems associated with this disease. Some DNA sequences are transcribed into RNA but are not translated into protein products—such RNA molecules are called non-coding RNA . In some cases, these products fold into structures which are involved in critical cell functions (e.g. ribosomal RNA and transfer RNA ). RNA can also have regulatory effects through hybridization interactions with other RNA molecules (such as microRNA ). Although genes contain all
14238-446: The similar molecule RNA instead of DNA as their genetic material. DNA normally exists as a double-stranded molecule, coiled into the shape of a double helix . Each nucleotide in DNA preferentially pairs with its partner nucleotide on the opposite strand: A pairs with T, and C pairs with G. Thus, in its two-stranded form, each strand effectively contains all necessary information, redundant with its partner strand. This structure of DNA
14364-496: The single celled alga Acetabularia . The Hershey–Chase experiment in 1952 confirmed that DNA (rather than protein) is the genetic material of the viruses that infect bacteria, providing further evidence that DNA is the molecule responsible for inheritance. James Watson and Francis Crick determined the structure of DNA in 1953, using the X-ray crystallography work of Rosalind Franklin and Maurice Wilkins that indicated DNA has
14490-441: The strands are separated, new partner strands can be reconstructed for each based on the sequence of the old strand. This property is what gives DNA its semi-conservative nature where one strand of new DNA is from an original parent strand. Although the structure of DNA showed how inheritance works, it was still not known how DNA influences the behavior of cells. In the following years, scientists tried to understand how DNA controls
14616-564: The study of inheritance in his inaugural address to the Third International Conference on Plant Hybridization in London in 1906. After the rediscovery of Mendel's work, scientists tried to determine which molecules in the cell were responsible for inheritance. In 1900, Nettie Stevens began studying the mealworm. Over the next 11 years, she discovered that females only had the X chromosome and males had both X and Y chromosomes. She
14742-536: The tryptophan synthesis process. Differences in gene expression are especially clear within multicellular organisms , where cells all contain the same genome but have very different structures and behaviors due to the expression of different sets of genes. All the cells in a multicellular organism derive from a single cell, differentiating into variant cell types in response to external and intercellular signals and gradually establishing different patterns of gene expression to create different behaviors. As no single gene
14868-482: The twenty possible amino acids in a protein or an instruction to end the amino acid sequence ; this correspondence is called the genetic code . The flow of information is unidirectional: information is transferred from nucleotide sequences into the amino acid sequence of proteins, but it never transfers from protein back into the sequence of DNA—a phenomenon Francis Crick called the central dogma of molecular biology . The specific sequence of amino acids results in
14994-545: The two is responsible for inheritance. In 1928 , Frederick Griffith discovered the phenomenon of transformation : dead bacteria could transfer genetic material to "transform" other still-living bacteria. Sixteen years later, in 1944, the Avery–MacLeod–McCarty experiment identified DNA as the molecule responsible for transformation. The role of the nucleus as the repository of genetic information in eukaryotes had been established by Hämmerling in 1943 in his work on
15120-437: The way traits are handed down from parents to offspring over time. He observed that organisms (pea plants) inherit traits by way of discrete "units of inheritance". This term, still used today, is a somewhat ambiguous definition of what is referred to as a gene. Trait inheritance and molecular inheritance mechanisms of genes are still primary principles of genetics in the 21st century, but modern genetics has expanded to study
15246-532: The work of the Augustinian friar Gregor Mendel in the mid-19th century. Prior to Mendel, Imre Festetics , a Hungarian noble, who lived in Kőszeg before Mendel, was the first who used the word "genetic" in hereditarian context, and is considered the first geneticist. He described several rules of biological inheritance in his work The genetic laws of nature (Die genetischen Gesetze der Natur, 1819). His second law
15372-518: Was able to conclude that sex is a chromosomal factor and is determined by the male. In 1911, Thomas Hunt Morgan argued that genes are on chromosomes , based on observations of a sex-linked white eye mutation in fruit flies . In 1913, his student Alfred Sturtevant used the phenomenon of genetic linkage to show that genes are arranged linearly on the chromosome. Although genes were known to exist on chromosomes, chromosomes are composed of both protein and DNA, and scientists did not know which of
15498-634: Was first mentioned in 2008. The number of manuscripts referring to RNA-Seq in the title or abstract (Figure, blue line) is continuously increasing with 6754 manuscripts published in 2018. The intersection of RNA-Seq and medicine (Figure, gold line) has similar celerity. RNA-Seq has the potential to identify new disease biology, profile biomarkers for clinical indications, infer druggable pathways, and make genetic diagnoses. These results could be further personalized for subgroups or even individual patients, potentially highlighting more effective prevention, diagnostics, and therapy. The feasibility of this approach
15624-591: Was first observed by Gregor Mendel, who studied the segregation of heritable traits in pea plants, showing for example that flowers on a single plant were either purple or white—but never an intermediate between the two colors. The discrete versions of the same gene controlling the inherited appearance (phenotypes) are called alleles . In the case of the pea, which is a diploid species, each individual plant has two copies of each gene, one copy inherited from each parent. Many species, including humans, have this pattern of inheritance. Diploid organisms with two copies of
15750-421: Was particulate, not acquired, and that the inheritance patterns of many traits could be explained through simple rules and ratios. The importance of Mendel's work did not gain wide understanding until 1900, after his death, when Hugo de Vries and other scientists rediscovered his research. William Bateson , a proponent of Mendel's work, coined the word genetics in 1905. The adjective genetic , derived from
15876-529: Was the inheritance of acquired characteristics : the belief that individuals inherit traits strengthened by their parents. This theory (commonly associated with Jean-Baptiste Lamarck ) is now known to be wrong—the experiences of individuals do not affect the genes they pass to their children. Other theories included Darwin's pangenesis (which had both acquired and inherited aspects) and Francis Galton 's reformulation of pangenesis as both particulate and inherited. Modern genetics started with Mendel's studies of
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