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restriction enzymes
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Terms in this set (103)
restriction enzymes
- enzymes that fragment the genome at specific places
- recognizes a specific sequence of bases anywhere within the genome
- cuts sugar phosphate backbones of both strands
restriction sites
- usually 4-8 bp of double stranded DNA
- often palindromic
blunt ends
restriction enzyme cuts are straight through both DNA strands at the line of symmetry
sticky ends
restriction enzyme cuts are displaced equally on either side of line of symmetry, ends either have 5' or 3' overhangs
4^n
formula for restriction fragment length where n is the number of bases in the restriction site
(genome size)/(fragment length)
number of fragments made by the restriction enzyme
small restriction enzyme
more fragments are made
large restriction enzyme
fewer fragments are made
gel electrophoresis
distinguishes DNA fragments according to size; compared to smaller genomes, larger genomes will have smear/more bands in the gel when digested by restriction enzyme
agarose gel
noncovalent associations of agarose; suitable for separating larger fragments up to 20kb
acrylamide gelcovalent bonding between polymers; suitable for separating smaller DNA fragments less than 1 kbgenomic cloningA piece of DNA digested with restriction fragment and taken from the genome of a cell or animal, inserted into a bacteriophage or other cloning vector and cloned to make librariesgenomic librarylong lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector; represents ALL REGIONS OF DNA equally, including intronsorigin of replication, selectable marker, polylinker with multiple restriction sitesvector componentscDNA cloning- produces DNA clones from mRNA sequences mimicking retroviruses - mRNAs sorted out from the rest of RNA by their poly A tails - use reverse transcriptase to create hybrid cDNA-mRNA molecules - denature the mRNA and cDNA strands and eliminate the mRNA transcript - use DNA pol and dNTPs to make a complementary cDNA strand using the first strand as templatecDNA libraryA limited gene library using complementary DNA. The library includes only the genes that were transcribed in the cells examined; ONLY EXONSSanger sequencing- creates sequencing reads of 800-900bp - involves chain terminating dideoxyribonucleotides with fluorescent tags --> ratio 1ddNTP:300dNTP important - labelled fragments are electrophoresed in special polyacrylamide gel that separates DNA fragments that differ in size by 1 NTtoo little ddNTPresults in long fragments all stuck at the top of the gel with no separationtoo much ddNTPresults in premature termination and short fragments all stuck at the bottom of the gel with no separationNext Generation Sequencing (NGS)- sequences millions of fragments simultaneously per run - not running sample through gel, not using ddNTPs - using reversible 3' hydroxyl block nucleotides - sequence length is short, 150-300NT, but can sequence more fragments and generate longer sequence reads - small fragments produced at every nucleotide incorporation, more accurate, doesn't rely on band quality separationthe human genome- exome (exon part of genome) is 1-2% - mostly intronic DNA and junk DNA - most DNA is repetitive; multicopy tandem repeats and transposable elementsgene-rich regions- Chromosomal regions that have many more genes than expected from average gene density over entire genome - Class 3 major histocompatibility complex (MHC) contains 60 genes within a 700kb region, most _____ _______ _________ of human genomegene familiesgroups of genes closely related in sequence and functiongene duplication and divergenceThe generation of extra copies of a gene in a genome over evolutionary time. A mechanism by which genomes can acquire new functions; evolveprotein domains- often encoded by exons - sequence of amino acids that fold into functional units - many genes are composed of multiple exons that encode discrete _______ ________domain architecturescreated by the shuffling, addition, or deletion of exons during evolution that creates new genes whose protein products have novel ______ _____________exon shufflingThe variation in the patterns by which RNA may produce diverse sets of exons from a single gene' over evolution has produced different transcription factors with distinct domainshomeodomainA conserved sequence of 60 amino acids used in the binding to DNA. Usually found in transcription factors, it is used to express genes that are related, more specifically in development to make tissues associated with one another.orthologous genesgenes in two different species that arose from the same gene in the common ancestor, usually retain same functionparalogous genesarise by duplication within the same species, often refers to members of a gene familysingle nucleotide polymorphism (SNP)- single base pair variation in the genome - 1 per 1 kb, most common type of genetic variant - detected by PCR, sequencing and ASO microarrays - allows tracing of evolutionary history; can be detected/sequenced, mappable and used as lociancestral alleleAllele carried by last common ancestor of two species (seen in the chimpanzee and one of the humans)derived allelethe changed nucleotide, not inheritedIndel/DIP- genetic variation caused by insertion or deletion - 1 per 10 kbsimple sequence repeats (SSRs or Microsatellites)- sequences of 1 to a few bases that are repeated in tandem less than 10 to more than 100 times - 1 per 30 kb - most common repeating units are 1, 2, or 3 base sequences - most occur outside the coding region and have no effect on phenotype - caused by spontaneous rare events from mispairing or stuttering during DNA replication - short repeated sequence gets expandedslippage of DNA pol- causes SSRs/microsatellites - DNA pol pauses at newly synthesized strand and slips, two triplet repeats fold and pair with each other, polymerization continues and 2 additional triplet repeats more than original are incorporatedcopy number variation (CNV)- tandem sequence repeats that are more than 10 bp long - 1 per 60kb - produced by unequal crossing over during meiosis 1 from misalignment - uncommon, most are inherited and not new mutationsdetecting microsatellites by PCR- PCR amplifies a target region of DNA - unique DNA flanks the microsatellite repeat, use to design PCR primers - primers allow DNA pol to generate new strands of DNA complementary to both strands of genomic DNA between the primers - denature the original template from new strands - gel electrophoresis can distinguish small variations in the size of a locus and separate based on size - each allele appears as a specific band of DNAAllele-specific oligonucleotides (ASOs)short oligonucleotides that hybridize with alleles distinguished by a single base difference; attached to a solid support; at specific temperature DNA probe will hybridize only to ASOs that match the probe perfectlyhaplotypesA group of alleles that tend to be inherited as a unit due to their closely spaced loci on a single chromosome.positional cloning- obtaining information about the location of the disease gene by finding polymorphic loci to which the mutation is genetically linked - issues: the phase problem, noninformative crosses, small sample sizeLaw of odds (LOD)- used to determine whether the data are sufficient to conclude with confidence that a disease gene and a DNA marker are genetically linked - to be linked, LOD>3chromosomal rearrangements- add or remove base pairs: deletions and duplications - relocate chromosomal regions without changing number of base pairs: inversions and reciprocal translocation - caused by DSB and NHEJ and abnormal crossing overdeletions- Mutation involving the removal of one or more nucleotide pairs from a gene - no recombination can occur within a __________ loop - map distances in heterozygotes will be shorter than expected because fewer crossovers can occur - homozygosity is often lethal or harmful - can uncover a recessive mutation = pseudodominancemutant gene must lie inside deletionheterozygote is mutant phenotypemutant gene must lie outside deletionheterozygote is wildtype phenotypeduplication- important in evolution; new functions arise as a process of _________ and divergence - most have no phenotypic consequences but large ones can be deleterious - unequal crossing over can increase or decrease copy numbertandem duplicationsthe repeated copies lie adjacent to each other in the same or reverse ordernon-tandem duplicationsthe copies of the region are not adjacent to each other and may lie far apart on the same or different chromosomespericentric inversioncentromere is within the inverted segment; breaks occur surrounding centromereparacentric inversioncentromere is not within the inverted segmentinversions- pericentric or paracentric - homologous regions in __________ regions can still pair and undergo crossover - formation of __________ loop allows tightest possible alignment of homologous regions - crossing over within the loop produces abnormal recombinant chromatidspericentric inversion crossover- each recombinant chromatid has a centromere, but each will be genetically unbalanced - zygotes formed from union with normal gametes and these will be nonviableparacentric inversion crossover- one chromatid lacks a centromere and the other has 2 centromeres - zygotes formed from union of normal gametes with these will be nonviablecrossover suppression- only gametes that did not recombine within inversion loop are viable - in inversion heterozygotes, no viable offspring produced - makes balancer chromosomesbalancer chromosomes- have multiple, overlapping inversions - have a dominant, visible, recessive lethal marker - useful as a genetic tool because no recombination will occurreciprocal translocation2 nonhomologous chromosomes exchange parts, no loss of genetic material; usually don't result in mutant phenotype; may result in decreased fertilitytranslocation homozygotesif breakpoints don't affect gene function, no genetic consequence; chromosomes segregate normally during meiosistranslocation heterozygotes- the 2 haploid sets of chromosomes carry different arrangements of DNA - chromosome pairing during prophase 1 is maximized by formation of cruciform structure - 3 types of segregationalternate segregation- produces balanced gametes and viable progeny - N1 + N2 and T1 + T2adjacent-1 and adjacent-2 segregation- produces unbalanced gametes with large duplications and deletions = lethaladjacent 1N2+T1 and N1+T2adjacent 2N1+T1 and N2+T2 less frequent, caused by nondisjunctionpseudolinkagethe appearance of linkage of two genes on translocated chromosomesFluorescence in situ hybridization (FISH)A procedure using a fluorescence-labeled probe to detect specific nucleotide sequences within intact cells attached to a microscopic slidechromosome paintinguse of differentially labeled, chromosome-specific DNA strands for hybridization with chromosomes to label each chromosome with a different colorRNA polymerase IIin eukaryotes transcribes genes that encode proteinscis acting elementspromoters and enhancerstrans acting elementstranscription factors: basal factors, activators, and repressorspromoters- position and orientation is critical; identifies transcriptional start point - usually directly adjacent to the gene - necessary for transcription to occur - TATA box 25-35 NT before transcription start site, AT rich sequence - bind RNA pol and allow basal level of transcriptionenhancers- A DNA sequence that recognizes certain transcription factors that can stimulate basal level of transcription. - position and orientation is flexible; can be located either 5' or 3' to transcription start site - intervening DNA bends so it can come together with promoterdetection of ehancers- construct a recombinant DNA molecule that has a possible enhancer sequence fused to reporter gene (like GFP) - if it is an enhancer, should see robust expression of GFP - can generate transgenic organism that has the recombinant DNA in specific tissuestranscription factors- sequence specific DNA binding proteins - bind to promoters and enhancers, recruit proteins to influence transcription - 3 types: basal factors, activators, repressorsbasal factors- transcription factor that binds to promoters in an ordered fashion - TATA binding protein (TBP) binds to TATA box and recruits TBP associated factors (TAFs) - RNA pol II binds to TAFs and initiation complex is associatedmediator- Complex of proteins that is one of the components of the basal transcription apparatus. - doesn't bind DNA directly, bridges RNA pol II at the promoter and activator or repressor at the enhancer - recruits proteins to the basal promoter; mediates interactions between promoter and enhanceractivator- transcription factor that binds to the enhancer and activates transcription from the basal promoter in 2 ways: - stimulate recruitment of basal factors and RNA pol II to promoters --> causes looping of DNA - recruit coactivators to open chromatin structure (HAT, HMT)structure of activatorsDNA binding domain, activation domain, dimerization domainDNA binding domainpart of the activator that binds to a specific enhancer; examples are Helix-turn-helix and Zinc Finger; fit within major groove of DNAActivation domainpart of the activator that binds to other proteins (basal factors or coactivators); less well defined structural elements compared to DNA binding domainDimerization domainsome activators have domain that allows them to interact with other proteins and allows for increased specificity; homodimers and heterodimers; example is Leucine zipperrepressors- transcription factor that suppresses transcription initiation by binding to enhancer and recruiting corepressors - indirect or directdirect repression- prevent binding of basal factors at promoter or close chromatin - repressors can recruit corepressors that directly prevent RNA pol II complex from binding promoter - repressors can recruit corepressors that close chromatin (HDAC, histone demethylase, HMT)indirect repression- interferes with the function of an activator - competition due to overlapping binding sites for activators and repressors - repressor binds to activation domain (quenching) - binds to activator and keeps it in cytoplasm (cytoplasmic sequestering) - binds to activator and prevent homodimerization; which creates inactive complexsteroid hormone receptor- has DNA binding motif (zinc finger), dimerization domain, activation domain, and ligand binding domain - allosteric interactions: binding of hormone causes protein to be able to bind target sequence due to conformational changemax protein- helix loop helix DNA binding protein - present in all cells - can form homodimers or heterodimers with Myc and MadMad protein- helix loop helix DNA binding protein - present in resting cells - can only form heterodimers with Max - contains repression domainMyc protein- helix loop helix DNA binding protein - not transcribed in resting cells, expressed when cells about to divide - can only form heterodimer with Max - has activation domainChIP-Seq (chromatin immunoprecipitation sequencing)- tool for finding all target genes of a particular transcription factor within the entire genome of a particular type of cell - crosslink DNA and protein component of chromatin - fragment DNA randomly - allow antibody specific to protein of interest to bind to fragment - purify complex with antibody, protein of interest, and DAN fragments - Sequence DNA to get the sequence of the targets recognized by the specific proteinEMSA (electrophoretic mobility shift assay)-Run DNA or RNA on gel, followed by DNA with potential binding partner. Look for shift on gel - the shift results from an interaction between DNA and protein, which forms a larger complex that moves more slowly on the gel in comparison to the control. This indicates that there is a protein-DNA interaction that should be further analyzedinsulators- sequences located between an enhancer and a promoter - block access to the promoters - human ________ bind CTCF proteins to form TADsTAD (topologically associated domain)- a fundamental structural unit of the genome that guides regulatory elements to their cognate promoters; separated by insulators - enhancers activate promoters located in the same loop; cant regulate a different looppolydactyly- caused by deletion of TAD boundary between EPH4A and IHH genes - usually IHH transcription is off because of insulator between the TADs - deletion of insulator allows EPH4A enhancer to activate IHH promoter in addition to EPH4A promoterchromatin conformation capture- used to detect TADS - chemicals are used to covalently cross-link proteins and DNA in close proximity within chromatin - DNA is fragmented and DNA ligase joins ends to form chromatin circles - high throughput methods used to define TADsSxl geneacts as a switch that selects the pathway of sexual development by controlling splicing of the dsx transcript in a female-specific fashion; only produced in female drosophila with 2 X's; alternatively slices Tra, Dsx, and Fru4E-BP1- binds to initiation factor elF4E (bound to 5' cap) - prevents initiation of translation by inhibiting elF4E from binding to PABP - nutrient rich environment: phosphorylated so it is inactive and translation can occur - nutrient rich environment: boundsiRNA- small interfering RNAs - targets are nascent transcripts of chromosomal regions designed to become heterochromatin - blocks translation/destabilize mRNAs - recruits histone-modifying enzymes to DNA resulting in heterochromatin formation - form RISC complexes with Argonaut proteinspiRNAs- targets transposable element transcripts and promoters - degrades transposable element - facilitates histone modifications that inhibit transposable element transcription - makes complexes with Piwi proteinsmiRNA- small mRNA that binds to sequences in the 3' UTR of target RNA and blocks translation - 20-30NTDroshaexcises stemloop from pri-mRNA to create pre-mRNADicerafter pre-miRNA is exported from nucleus to cytoplasm, processes it to leave just the duplex miRNARISC (miRNA induced silencing complex)- one strand of the duplex miRNA is incorporated into the complex which can act as guide miRNAtarget mRNA is degradedperfect complementarity between mRISC and target mRNAtranslation of target mRNA is repressedimperfect complementarity between mRISC and target