Restriction-enzyme techniques can recombine nucleic-acid sequences {recombinant DNA}|.
Experiment assays can analyze physical substances {sample, DNA}. Samples come from subjects.
Diluting main sample {aliquot}| creates secondary samples.
Samples {parent sample} can make aliquots.
Sample information is in databases {logging}.
Most experiments test samples by the same method more than once {replicate, sample}|.
Techniques {amplification, DNA} {DNA amplification} can increase number of DNA-fragment copies. In vivo amplification amplifies repeat sequences in fragile X syndrome. In vitro amplification uses cloning or polymerase chain reactions.
purposes
Amplified DNA indicates mutations, translocations, viral and bacterial infections, sex, genealogy, living and extinct species differences, and forensic identification.
DNA synthesis
To make probes or primers, first blocking-group covers 5' or 3' hydroxyl groups of two nucleotides. Then other two hydroxyl-groups react to make phosphodiester bonds. Then acid or base removes blockers. Process repeats to elongate chain.
After restriction-enzyme digestion, DNA fragments can amplify, and relative lengths indicate polymorphisms {Amplified Fragment Length Polymorphism} (AFLP).
After electrophoresis, DNA fragments flow in transparent tubes {cuvette}|, and lasers excite dyes.
Two complementary DNA or RNA single strands can form a double-stranded molecule {hybridization, DNA}|. Two nucleic-acid strands can pair by hydrogen-bonding A and T, or A and U in RNA, and C and G. DNA or DNA and RNA complementary-strand nitrogenous bases can have hydrogen bonding.
arrays
All array spots have hybridization, as cartridge holds array in position. Hybridization measurement depends on dye sensitivity and dynamic range. If array spots are close together, they can cross-hybridize. Evaporation also causes problems, so arrays have humectants, lids, or dewpoint controls. Spotting pins must be 0.2 mm small and clean. Alternatives to spotting include acoustic focusing, multi-nozzle piezoelectric jets, and continuous solid pin spotting. Probes, cDNA arrays, and oligonucleotide arrays are alternative hybridization methods.
Methods {polymerase chain reaction}| (PCR) can make many DNA-sequence copies using heat-stable polymerase, 20-base primers complementary to + strand at one sequence end, and 20-base primers complementary to - strand at other end. Synthesized strands are additional templates, so process doubles copies each primer-annealing, strand-elongation, and dissociation cycle.
purpose
PCR can detect defined sequences in DNA samples. PCR can make stutter bands and add bands resulting from extra nucleotide addition by Taq polymerase.
mRNA amplification
DNA has small amounts, but mRNA has much larger amounts. First, reverse transcription converts mRNA to cDNA and then PCR amplifies cDNA (RT-PCR).
DNA amplification
Machines heat DNA double helix to 94 C for several minutes to make single-stranded DNA. Solution contains DNA polymerase from heat-tolerant organisms and the four bases.
When temperature lowers to 30 C to 65 C for 30 seconds, 20-nucleotide primer DNA binds to DNA, outside region to copy. One primer is for 5' strand, and one primer is for 3' strand. Annealing puts complementary 20-base primer at both ends.
Machines raise temperature to 72 C for some minutes, to allow DNA polymerase and bases to extend both primers beyond other primer region. Now both double-helix molecules have primer on one end and extend beyond other primer on other end. Elongating both strands uses heat-stable DNA polymerase, which synthesizes DNA.
Machines heat DNA to 94 C for several minutes again to extend same primers through other primer at strand ends. Now all synthesized-strand lengths are the same, from one primer through other primer. There are now four DNAs.
Cycles make twice as many DNA strands, and process uses new and old strands again, making chain reactions. Repeating process 30 to 60 times makes millions of copies.
primers
Primers can be genome repetitive sequences, such as Alu repeats. Alu repeats are 300 bases, but smaller region varies little in humans. Alu repeats are in both directions.
primers: nested
After one PCR, second primer that binds inside copied sequence {nested primer} can amplify shorter sequences.
primers: concentration
If one primer has high concentration and one has low, system makes mostly single-stranded DNA {asymmetric PCR}, with no chain reaction.
contamination
Contamination with wrong DNA is common. Negative controls make sure correct DNA amplifies.
Sequence tags {adapter sequence} are in probes.
Single-stranded DNA sequences {oligonucleotide}| {oligo} can have less than 61 bases.
To add deoxyribonucleotides to nucleic acid by DNA polymerase requires oligonucleotides {primer, DNA}|.
Short RNAs or single-stranded DNAs {probe, DNA} {DNA probe} can detect complementary base sequences by hybridization. Probes have 25 to 60 bases and can have 3'-hexyl-amine. Probes attach to last 1500 base pairs closer to transcript 3' ends, where genes have unique short DNA regions. Bacteria and yeast genes have unique primers. Higher organisms have three million different expressed sequence tags (EST).
process
High-concentration purified probes are in 96, 384, or 1536 wells on plastic microtiter plates. Robots take probes from microtiter plate to make same number of spots on glass slides, one slide for each RNA sample to test. Multiple probes test each gene.
Nucleotide sequences {zipcode} can attach to molecules to allow probe complementary nucleotide sequence {zipcode complement} to hybridize.
Genomes can have known sequences {DNA sequencing}. H. influenzae has 1.8 million bases and 800 genes. E. coli has 4.5 million bases and 2000 genes. Saccharomyces yeast has 12 million bases and 6000 genes. P. falciparum has 30 million bases and 6500 genes. Caenorhabditis elegans roundworm nematode has 100 million bases and 10000 genes. Arabidopsis thaliana weed has 120 million bases and 20000 genes. Drosophila melanogaster fruitfly has 165 million bases. Fugu rubripes is zebrafish. Mus musculus mouse has 3000 million bases. Humans have 3500 million bases and 30000 genes.
methods
Sequencing {plus-minus method} can elongate DNA sequences using DNA polymerase.
Sequencing {Maxam-Gilbert sequencing method} can chemically degrade labeled, short DNA chains at G, C, A and G, and T and C to make fragments and then electrophorese all four, separately or together, to separate by size.
Sequencing {Sanger sequencing method} can elongate DNA chains and randomly terminate them with nucleotides that cannot bind to next ribose, using A, G, T, and C 2',3'-dideoxynucleoside triphosphates, and then electrophoreses all four, separately or together, to separate by size.
Mixing differently labeled clones {multiplex DNA sequencing} allows easier processing. Labels identify clones.
DNA-fragment ends can have fluorescent dyes, which respond to laser light. Dye terminator attaches to nucleotide 3' ends. Dye primer attaches to 5' ends. Longer fragments have higher signals. Fragments separate by electrophoresis in gels {horizontal ultrathin gel electrophoresis} (HUGE) or capillary tubes {capillary gel electrophoresis}. Because charges are equal, fragments leave gel or tube by size.
Scanning tunneling electron microscopes can sequence DNA strands by wand distance needed to maintain constant voltage. They can sequence 40000-base DNA fragments one base at a time. Exonuclease removes end nucleotide. Shining laser light and recording with photomultiplier reads nucleotide type.
Gas or liquid mixtures can separate using gas chromatography, liquid chromatography, supercritical fluid chromatography, or capillary electrophoresis.
Computers overlap sequence information about DNA fragments {contig, sequencing} to build longer sequences, to map large fragments and then chromosomes.
Cluster hierarchies can look like tree diagrams {dendrogram, sequencing}.
If many DNA fragments insert into many bacteria, plate colonies can account for all DNA fragments {DNA library} from foreign organisms.
DNA analyses include comparing DNA sequences. Sequences of same gene from different organisms can align somewhat {homology, DNA}| {homologous sequences}.
Probes hybridize with clone and fragment regions. Overlapping DNA fragments hybridize to same probe. 20-base oligonucleotides can be probes to find overlapping DNA fragments. For clone ends, random sequences can be probes {sequence-tagged connector} (STC). Probes can have minor-groove binder to enhance exact hybridization, allowing shorter probes.
Processing identifies unique 200-base to 500-base sequences, with unique primers, from known locations {sequence-tagged site} (STS). Perhaps, clones share STS.
After DNA fragments exit capillary, plot {electropherogram} shows relative dye concentration versus time expressed as frame number. In electropherograms, small peaks {pull-up peak} can appear under main dye peaks. Incorrect dyes, dye contamination, or capillary or fluid-property changes after spectral calibration can cause pull-up peaks.
In direct methods, mRNA-AAAAA + reverse transcriptase + Oligo-dT Primer + dNTPs + Cy3 and Cy5 or SymJAZ dye-dNTP -> Dye-cDNA {DNA labeling}.
In random priming methods, mRNA + reverse transcriptase + T7-T20-24 + MuLV -> DNA/RNA + hydrolysis -> cDNA first strand + Bst DNA polymerase + ligase + pN8-9 -> T7-ds cDNA + dye-UTP + T7 RNA polymerase + IVT -> labeled cRNA.
In RNase H methods, mRNA + reverse transcriptase -> DNA/RNA + RNase H -> DNA/RNA + DNA polymerase + ligase -> T7-ds cDNA + dye-UTP + T7 RNA polymerase + IVT -> labeled cRNA.
purposes
DNA labeling can measure labeled-cRNA dye incorporation, reverse-transcriptase conversion, fluorescence-specific activity, minimum RNA, maximum RNA, IVT amplification, total amplification, and length.
controls
Control reagents aid spot finding, image analysis, and signal quantification. Array probes monitor spotting, labeling, hybridization, printing, attachment, and features. Labeling controls monitor enzyme activity, target stability, and dye incorporation during labeling protocols. Hybridization controls monitor mixing, stringency, and washing during array-hybridization protocol. Printing and attachment controls monitor array manufacturing, probe attachment, and lot-to-lot variability. Feature controls normalize signal variability.
DNA analyses include finding sequences longer than cloned DNAs by looking for their overlaps. First clone can screen whole-genome clone library for overlapping sequences. If overlap, second clone can screen, and so on, to build longer and longer sequences {chromosome walking}.
DNA sequencing can use modified shotgun methods {polony sequencing}.
DNA-fragment mixtures can separate fragments by lengths {fragment analysis}.
Electrophoresis gels have band sequences {lane}, starting from top sample bands.
Electrophoresis separates eluted DNA fragments, to compare peak separations, spectral separations, and spatial separations {fragment separation}.
Compounds less soluble in stationary phase elute faster {retention time, elution}.
DNA analyses can separate cell RNAs or mRNAs on gels and transfer gel-band contents to filters, where RNAs can hybridize to known RNA or cDNA sequences {Northern blotting}. Northern blotting compares mRNAs to cDNAs to study gene expression.
DNA analyses can separate DNA fragments on gels and transfer gel-band contents to filters, where DNA fragments can hybridize to known DNA sequences {Southern blotting}. Southern blotting can detect gene rearrangements that make antibodies and T-cell receptors. It can detect disease-caused gene rearrangements and deletions. It can detect related genes in organisms and homologous genes from different species. It can detect mRNA-splicing-caused intron removal and exon use. It can detect mRNA splicing to make alternative proteins. It can detect nested genes.
DNA transcription makes tRNA, rRNA, and mRNA {gene expression}|.
purposes
Gene expression studies gene functions, regulation, and interactions. Hybridization measures gene expression for gene discovery, gene identification, biochemical pathways, and disease mechanisms.
probes
Human-genome arrays have probes for all genes. Human-transcriptome arrays have probes for all transcripts. SNP arrays have all SNPs. Arrays can have immune, toxicity, or cancer-gene probes.
RNA or single-strand DNA oligonucleotides {antisense RNA}|, complementary to cell mRNAs, can bind to mRNA and prevent gene expression. Antisense RNAs can be in vectors, or techniques can inject them into cells.
300-base to 500-base sequences {expressed sequence tag} (EST) are specific to expressed gene regions, typically at 3' ends. ESTs map gene chromosomal locations from several tissues, recover corresponding gene sequences by electronic-database similarity searching, and retrieve complete cDNA clones for further analysis. Whole-genome shotgun sequencing using EST assembly reduces redundancy and creates longer consensus sequences.
For gene-probe spots and dyes, software calculates ratios {expression ratio} {gene expression ratio}: normalized expression level divided by normalized expression level for control gene. Average expression ratio is 1. If expression ratio is greater than or equal to 2 {up-regulated, expression} or less than or equal to 0.5 {down-regulated, expression}, genes have significant expression {differentially expressed}. Expression-ratio base-two logarithm averages 0, is +1 if expression ratio is 2, and is -1 if expression ratio is 0.5.
Gene similarity measures can be distances {gene distance} between expression vectors in expression space.
metric
Distances can have metrics {metric distance}. Distance can always be positive. Distance between point and itself can always be 0. Euclidean distance between two points can always be less than or equal to sum of distance from first point to third point and distance from third point to second point {triangle rule}.
metric: Euclidean distance
Euclidean distances can be differences in point coordinates. Euclidean distances are metric.
semi-metric
Distance measures {semi-metric distance} can be always positive and have distance between point and itself always zero, but not obey triangle rule.
To emphasize variation amounts, especially for timed experiments, methods {scaling method} can reduce large expression-ratio values by changing expression-ratio range. Scaling can set average logarithm to zero {mean centering}, by subtracting baseline value. Scaling can adjust logarithm range to -1 to +1. Scaling can normalize expression-vector magnitudes to 1.
Gene-expression technologies can use cell extracts from different tissues, same tissues under different conditions, or same tissues under same conditions, over time sequences {serial analysis of gene expression} (SAGE) {expressed RNA}. From RNA, two cell extracts from same tissues under same conditions can make first-strand cDNA labeled with fluorescent dye, one with Cy3 and one with Cy5. Purified labeled cDNA solution soaks slides at optimum temperatures for times. Robots measure slide-spot probe-DNA and labeled-cDNA hybridization.
Compared to control level, genes can have less expression {down-regulated, gene}. Less expression under same conditions indicates similar biological functions.
Compared to control level, genes can have more expression {up-regulated, gene}. More expression under same conditions indicates similar biological functions.
Artificial situations {experiment} can test hypotheses or answer questions. Genomics experiments use one or more assays, samples, and markers.
Arrays can have random sets of spots with various concentrations and known green-intensity vs. red-intensity ratios {Random Ratio Dilution series test} (RRD). Automated spot finding works at the 85% level. Variation coefficient {coefficient of variance} (CV) is less than 20%.
Lasers can fluoresce microarray to read sample results {reader, microarray} {microarray reader} {scanner, microarray} {microarray scanner}. Displays can zoom, track, and normalize arrays or array sets. Dual red/green lasers need constant laser-spot size and large field depth. Scanning simultaneously minimizes spatial crosstalk. Microarrays are in automatic loaders to maintain positions.
Control samples {standard sample} calibrate instruments or methods.
Oligonucleotides attach to plates {array} in rectangular patterns, to test one sample for hybridization.
plates
Plates can be silicon chips {DNA chip}, plastic blocks with small wells {microarray, plate}, optical-fiber tips {bead array}, or glass slides {planar array}.
process
For example, plastic blocks have wells. Wells have reactive-chemical solutions to assay samples. Array probes samples by hybridizing oligonucleotides to sample RNAs or cDNAs. Reader detects hybridization amount using light. Statistical and comparative calculations follow.
Sample plates {master plate} stored in freezers can make daughter plates.
Master plates supply other plates {daughter plate}.
Arrays {microarray} can have 9x9-spot matrices at hundreds of positions, to test many genes against one or more test oligonucleotides, plus controls and fiducial-probes.
Plates {microtiter plate} can have small wells.
Sample plates contain pits {well, array} that can hold one or more samples.
DNA fragments inserted into host nucleic acids can replicate in host organisms {cloning}|.
hosts: bacteria
Plasmids can insert up to 1000 bases. 50,000-base bacteriophage viruses can infect bacteria and can insert up to 15,000 bases. 300,000-base bacterial artificial chromosome DNA can have all bacterial-chromosome functional regions. Cosmids can hold 45,000 bases between cos sites. Gene-product secretion is preferable to harvesting cells.
In bacteria hosts, eukaryote proteins do not fold properly. Foreign proteins can kill bacteria. Bacteria have no post-translation enzymes.
hosts: yeast
Gene-product secretion is preferable to harvesting cells. In yeast hosts, proteases can destroy generated proteins.
For yeast hosts, replicating nucleic acid can be yeast artificial chromosomes.
Two-micron-circle yeast plasmid has replication origin that makes many copies per cell cycle. Other plasmids that use autonomously replicating sequence, sometimes helped by centromere sequence, make one or two copies per cell cycle. Yeast plasmids {shuttle vector} can work in bacteria.
Yeast vectors {integrating vector} with no replication origin integrate gene into yeast genome.
hosts: plants
For plant hosts, replicating nucleic acid can be Ti plasmid.
hosts: insects
For insect hosts, replicating nucleic acid can be baculovirus. Insect cell cultures have high costs. Gene-product secretion is preferable to harvesting cells.
hosts: mammals
For eukaryotic hosts, replicating nucleic acid can be virus or retrovirus. Mammalian cell culture has highest costs. Gene-product secretion is preferable to harvesting cells.
DNA fragment
DNA fragments can come from foreign organisms by cutting chromosomal DNA into DNA fragments using restriction enzymes. DNA fragments can come from mRNA by making cDNA from mRNA using reverse transcriptase and then making double-stranded DNA from cDNA. Synthesis methods can synthesize DNA.
polylinker
DNA fragments have polylinkers added at both ends, to allow nested cuts by different restriction enzymes.
insertion
DNA fragments can link into replicating nucleic acids using restriction enzymes to cut both nucleic acids and then allowing recombination.
selection
After replicating nucleic acids go into hosts, agents kill hosts if they do not have protecting genes in replicating nucleic acids. For example, bacteria with no plasmids die, because plasmids have genes to protect against antibiotics.
DNA
Host cells that live have DNA fragments, for extraction or secretion. Hybridization can test extracted or secreted DNA for DNA fragments. DNA sequencing can test for DNA fragments. Antibody binding or direct protein assays can test extractions or secretions for DNA-fragment gene products.
Toxic changed or foreign genes destroy tissue {cell ablation, cloning}.
Organisms, cells, and molecules can duplicate {clone}|.
Bacteria {colony, bacteria}| can grow on media.
DNA analyses can cleave chromosomes by restriction enzymes to make DNA fragments, separate fragments by size, and use fragment overlaps to mark relative restriction-enzyme-site positions {restriction map}.
Blunt ends can become sticky by attaching DNA {linker} containing recognition sites to blunt ends and then cleaving with restriction enzymes.
DNA fragments inserted into replicating nucleic acids can have many possible restriction enzyme sites {polylinker}, to allow nested cuts by different restriction enzymes.
Inheritable DNA-sequence positions {marker}| are restriction-enzyme cutting sites, fragment-length polymorphisms, genes, minisatellite DNAs, or microsatellite DNAs. Markers have inheritance patterns.
Replicated nucleic acids have added genes {marker gene}, to indicate foreign-DNA insertion and DNA replication.
bacteria
Hosts with added antibiotic resistance genes make proteins that resist antibiotics, whereas hosts with no such genes die. Beta-galactosidase gene makes protein that metabolizes galactose and makes color. Hosts with no beta-galactosidase gene have no color.
yeast
Yeast can grow without leucine if they have LEU gene, without histidine if they have HIS3 gene, without lysine if they have LYS2 gene, without tryptophan if they have TRP1 gene, and without uracil if they have URA3 gene.
plants
Genes {beta-glucuronidase gene} {GUS gene} can make protein that makes glucuronides. Plants have no glucuronides, so E. coli GUS genes can be markers for plants. Firefly luciferase gene makes light. Luciferase genes can be reporter genes for plants.
mammals
Thymidine kinase (tk) gene makes protein that makes thymidine triphosphate {thymidylate}. Mammalian cells (tk-) can have no thymidine kinase gene, so thymidine kinase genes can mark cells (tk+). Aminopterin inhibits all other thymidylate synthesis pathways, so only thymidine kinase gene can make thymidylate.
drugs
G418 inhibits protein synthesis and causes cell death. Aminoglycoside phosphotransferase (APH) gene makes protein that inactivates G418.
Methotrexate inhibits dihydrofolate reductase and causes cell death. Methotrexate-resistant dihydrofolate reductase (DHFR) gene makes protein that resists methotrexate.
Hygromycin-B inhibits protein synthesis and causes cell death. Hygromycin-B-phosphotransferase gene makes protein that alters hygromycin-B.
Mycophenolic acid inhibits GMP synthesis and causes cell death. Xanthine-guanine phosphoribosyltransferase (XGPRT) gene allows GMP synthesis from xanthine.
9-beta-D-xylofuranoyladenine (Xyl-A) damages DNA and causes cell death. Adenosine deaminase (ADA) gene metabolizes Xyl-A.
Replicating nucleic acids can have added genes {reporter gene} that catalyze reactions used to report that promoters are working or not, for gene-expression or transcription-factor studies. For example, chloramphenicol acetyltransferase gene (CAT) reacts with chloramphenicol. Reporter genes are after promoters, to provide direct promoter-activity assays.
If electric fields make holes in bacterial membranes {electroporation}, plasmids can enter bacteria.
Plasmids can enter bacteria during short high-heat periods {heat shock}|, in concentrated calcium-chloride solution.
Lipid vesicles {liposome}| with DNA or protein can fuse with cell membranes and enter cells.
Replicating nucleic acids can go into host organisms to make different organisms {transformation, DNA}|. Plasmids can enter by heat shock or electroporation. Bacteriophages can infect bacteria naturally. Transforming prokaryotic cells has high success rate.
Soy, maize, and other organisms {genetically modified organism} (GMO) can have deliberate genetic changes by genetic engineering.
Replacing genes with bad genes {knockout gene} makes animals that lack proteins. Transgenes can insert into normal gene positions, causing gene-function loss and affecting development.
Genes can transfer into eukaryotic-cell genomes {transfection}|. Mice, plants, and yeast have only one transfection per thousand cells. DNA can go to cell nucleus but not enter genome, so gene expresses until DNA breaks down {transient expression}. Transfection takes time. Mammalian cell lines must be immortal. Cell culture requires many cells.
types
Inject DNA fragments into cell nucleus {microinjection}. Precipitate DNA fragments with calcium phosphate, so cell-culture cells absorb precipitated DNA by endocytosis. Make liposome lipid vesicles, with DNA inside, that can fuse with cell membranes and enter cells. Fire tungsten microbullets, with DNA fused to them, into plant cells, to penetrate cell wall.
types: virus
Viruses can transfect. Omitting coat proteins prevents virus formation, so cells do not die.
Monkey COS cells include most SV40-virus DNA and make T antigen, which binds to SV40 replication origin. Plasmids with SV40 replication origin can transfect COS cells. Vaccinia virus is large and can hold bacteriophage RNA polymerase. Plasmids with bacteriophage promoter can transfect cells and suppress cell mRNAs. Insect baculovirus DNA is large and can hold genes in coat-protein DNA.
types: retrovirus
Retroviruses can go into all mammalian cells. Retroviruses first place provirus DNA sequence in genomes and then make retroviral RNA. The next stage {packaging, virus} makes complete viruses by adding coat proteins. Then cells die and release viruses. For transfection, experimenters remove packaging genes from retrovirus {helper-free}, to prevent making complete viruses, so cells live.
Changed or foreign genes can enter mouse embryo cells {transgenic mice} at chromosomal positions. Transgenic-mice descendants have changed or foreign genes and have new proteins.
organism
Mammals have cell and tissue interactions, so testing requires whole organisms.
process: injection
SV40, Moloney murine leukemia virus (MoMLV), or mouse mammary tumor virus (MMTV) microinjection can put changed or foreign DNA into cells. Cloned-gene microinjection into fertilized egg pronuclei can put changed or foreign DNA into cells.
process: cell addition
Mice embryos can change by adding altered cells. Mouse blastocysts have inner-cell {embryonic stem cell, blastocyst} (ES cell) layers, which can culture with fibroblasts or with leukemia inhibiting factor to prevent further differentiation. Embryonic stem cells can uptake and insert genes by homologous recombination. Then ES cells go into mouse embryos.
marker
Neo gene resists G418. ES cells with neo gene resist G418 and live.
embryonic development
In embryos, tissue-specific regulators express changed or foreign genes in one tissue but not different tissues. If changed or foreign genes are toxic, they destroy tissue {cell ablation, toxin}. Ablated cells prevent subsequent tissue development, allowing embryo location and function tracking {cell lineage study}. Retrovirus with E. coli lacZ reporter can trace tissue differentiation and cell migration.
Chinese-hamster ovary (CHO) cells {transgenic tissue} can track transgenic effects. Mammary glands can express transgenes.
DNA or RNA sequences {cloning vector}| can contain DNA or RNA from other sources and can replicate in host organisms. Vectors include plasmids, phages, retroviruses, cosmids, baculoviruses, bacterial artificial chromosomes, and yeast artificial chromosomes.
Cauliflower-Mosaic-Virus promoters {35S promoter} can be in soy, maize, and other genetically modified organisms.
Vectors {Bacterial Artificial Chromosome} (BAC) derived from F-factor plasmids can clone 100,000-base to 300,000-base DNA fragments in Escherichia coli.
Cloning-vector plasmids {cosmid} can contain lambda-phage cos gene, infect E. coli, and clone DNA fragments up to 45,000 bases between phage-end cos sites.
Bacteria can have 5000-base circular DNAs {plasmid}| that can insert up to 1000 bases. Cells can have 10 to 200 independently replicating plasmids {relaxed-control plasmid}. Plasmids {stringent-control plasmid} can replicate together with bacterial chromosomes. Artificial plasmids can be cloning vectors.
For yeast hosts, replicating nucleic acids can be artificial DNA with all yeast-chromosome functional regions {yeast artificial chromosome} (YAC). YAC replicates like yeast chromosomes.
parts
Yeast artificial chromosomes contain autonomously replicating sequence, centromere (CEN), and telomeres.
gene size
YAC can hold 100,000 bases. Several YACs can undergo homologous recombination to create complete genes from fragments.
Yeast artificial chromosomes contain sequences {autonomously replicating sequence} (ARS) for replication.
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Date Modified: 2022.0225