Molecular Bio Chapter 3
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52 terms
Terms | Definitions |
|---|---|
 cant really obtain a sequence of more tha 750 bp; needs to be constructed from series of shorter ones | one major limitation of sequencing |
| shotgun approach | genome sequencing strategy in which the molecules are randomly broken into fragments, sequenced individually and then a computer is used ot search for overlaps and build up the mastr sequence-- good for small prokaryotic genomes but not so much for larger ones, can also lead to errors with many overlapping regions |
| genome map | for a large molecule with many tandem repeats, a _________ must be generated, providing a guide for the sequencing experiements by showing positions of genes and other features |
| whole genome shotgun method | same as the regular one, but this one takes into account the landmarks from the genome map to aid assembly of the master sequence --rapid way of creating a draft of a euk. genome |
| clone contig method | genome sequencing strategy in which the genome broken into manageable segments, about a few hundred KB or a few MB, short enough to be sequenced by the shotugn method more accurately--takes longer, but more accurate. ( Larger fragments ) |
| - genetic mapping - physical mapping | 2 categories of genome mapping |
| genetic mapping | based on the use of genetic tecniques to to construct maps showing the position of genes and other sequence features ona genome - tecniques include cross breeding, or pedigree analysis |
| physical mapping | uses molecular bio techniques to examine DNA molecules directly in order to construct maps showing the position of sequence features, including genes |
| alleles | to be useful in genetic analysis, construction of gene maps, a gene must contain at least 2 _____; but genes are not ideal, firstly not all of them are easily distinguished in order to be mapped, and also they are widely spaced apart in euk. genomes...other markers are needed |
| DNA markers - RFLP's - SSLP's - SNP's | mapped features that are not genes; like genes, they also need 2 alleles to be useful |
| RFLP's (restriction fragment length polymorphisms) | restriction enzymes cut DNA at specific sites, so that when treated with a specific enzyme, one should obtain the same bands every time..However some of these restriction sites are polymorphic, meaning that they have 2 forms...sometimes they have the cut site, other times they dont - result is that 2 adjacent restriction fragments remain linked together, resulting in a length polymorphism...run a gel and usually cant see the few polymorphic molecules, use southern hybrization & PCR to locate them with special primers that anneal to either end of the polymorphic site |
| SSLP's (simple sequence length polymorphisms) | arrays of repeat sequences that display length variations, because eachallele has a different number of repeat units. Can be multiallelic,unlike RFLPs, because each allele can have a number of different length variants 2 types: mini and microsatellites (microismore commonly used) |
| minisatellites or VNTR (variable # of tandem repeats) | a type of SSLP in which the repeat unit is up to 25 bp in length-- not spread evenly around hte genom,e but more likely found near the telomeric sequences, and also less efficiently typed by PCR, single it cannot accurately work with sequences > 300 bp, which they usually are |
| microsatellites or STR (simple tandem repeats) | a type of SSLP in which repeat units are shorter, typically 13 bp. More popoular as DNA markers because they are more evnely spread around the genome, and also because to type them, PCR is much more efficient being that they are shorter |
| SNP (single nucleotide polymorphism) | positions in a genome where some individuals have one nucleotide, ie C, while others have another nucleotide, i.e. G ..some of these give rise to the RFLPs ..occur from a point mutations--most are only biallelic, a third allele would have to arise from another mutation at that same point in a sec cell (improbable) |
| oligonucleotide hybridization analysis | short, ss DNA molecule, formed in a test tube, that only under perfect conditions will hybridize with another DNA molecule--if there is even one mismatch, it wil not hybridize --it can therefore discriminate between the 2 alleles of SNP ...this strictness is achieved by setting the temp at just below the melting temperature for the oligonucleotide. types include: 1) DNA chip technology 2) OLA |
| Tm | the ______ temperature of the olignucleotide- determinted by Tm= (4X GC) + (2X AT) , for an oligonucleotide 15-30 bp |
| DNA chip technology | a way to screen for SNPs by using a glass or silicon wafer containing many different oligonucleotides (high density microarray)...the DNA to be tested is labelled with a flourescent marker and pippeting onto the surface of the chip-hybridization is detected with a flourescent microscope --modified nucleotide substrates are used, ones that have to be light acitvated before they will attach to growing end |
| photolithography | a technique that uses pulses of light to construct an oligonucleotide from light activated nucleotide substrates |
| OLA (oligonucleotide ligation assay) nick= single strand break gap = ds break | makes use of 2 adjacent oligonucleotides that anneal adjacent to one another , with teh 3' end of one positioned exactly at the SNP...this will form a completely bp'ed structure if one version of the SNP is present, forming the ligation of the 2 oligonucleotides, but if the other allele is present, it cannot bp together, and there is a gap, hence no ligation product |
| ARMS test (the amplification refractory mutation system) | based on the same principle as the OLA, but this time, the test oligonuc. is one of a pair of PCR primers. If the primer can anneal to the SNP, then PCR produts are formed by Taq. Polymerase...if it cannot because the other allele is present , then no PCR products are formed |
| 1. alleles segregate randomly 2. pairs of alleles segregate independantly | Mendels 2 Laws |
| genentic mapping | linkage analysis is the basis for |
| partial linkage | If 2 genes are located close enough that sometimes they are inherited together and sometimes not, they are |
| bivalent, or tetrade | during Prophase I, each of the 2 homolougs line up to form a ______ before crossing over, or homologous recombination |
| recomination frequencies | 2 genes that are close will be separated less than 2 genes that are far away, assuming that crossing over can happen anywhere (wrong- recombination hotspots) .the frequency with which genes are unlinked by crossing over will be directly proportional to their distance apart on the chromosomes..._____ ______ are used to construct a physical distance map between 2 genes--but he was kinda wrong, did not take into accout recombination hotspots - linkage analysis does make generally accurate deductions about gene order, though |
| recombination hotspots | some regions of chromosomes that are more likely to be involved in a crossing over event--despite this , linkage analysis does make generally accurate deductions about gene order |
| 3:1 | indcative ratio of a linked pair of gene (treat like a monohybrid cross) |
| 9:3:3:1 | indicative ratio of an ulinked pair of genes-- if its not 3:1 or this one, then its notclear could be partially linked |
| (tryptophan) auxotroph | in transfer of a functional gene from a wild type to a recessive phenotype, the mutant bacterium that can survive only if provided with a nutrient that the wiltype doesnt need ..i.e. Trp+ ---> trp- , the trp- bacterium in this case being a "_______ ________"--cant synthesize tryptophan itself |
| 1. restriction mapping 2. FISH (flouresecnt in situ hybridization 3. STS (Sequence tagged site) mapping | Techniques for Physical Mapping |
| restriction mapping | in genetic mapping, RFLP's are used--but many restriction sites are not polymorphic at all...so we use this technique to map them. compare the fragment sizes produced when a fragment is digested with 2 different enzymes, like EcoRI and BamHI. |
| double restriction | digestion with 2 restriction endonucleases at the same time, to see if we can get more info for restriction mapping (a tecnique for physical mapping) |
| partial restriction | usually gives the info needed to complete a map, provides additional more complex fragments, that may still have uncute sites...suboptimal incubation temperature, or dont let the reaction run to finish |
| Label the molecule 5' end with a phsopahte isotope to visualize it , this way restriction sites without labels are "invisible", and sites can be calculated with relative distance from the molecules end | what can we also do to help us interpret partial digestions and create a restriction map? label the _______ |
| Use rare cutters i.e.- 1. only cuts once very 7-8 bp, instead of normal 6 bp 2. some cut only a rare DNA motifs, like a 5-CG-3 (which thanks to methylation of hte C, followed by deamination, gets truned often to 5'TG3' | How do we use restriction mapping for DNA bigger than its upper limit of 50 kb? (poor separation in standrd. gel electrophoresis for molecules >50 kb) |
| OFAGE (orthogonal field alteration gel electrophoresis) | DNA runs zig zag, diagonally, gives better resolution than standard gel electrophoresis --shorter molecules realign more quickl every time the field switches, migrating more rapidly down the gel |
| CHEF (contour clamped homogenous electric fields) | related to OFAGE, hexagonal shaped gel |
| FIGE (field inversion gel electrophoresis) | related to OFAGE-- runs it up and down, inverted field to separate out the bigger DNA fragments |
| optical mapping, main techniques include: - gel stretching - molecular combing | when restriction sites are directly located by looking at the cut DNA molecules under a microscope, first applied to large DNA fragments in BAC and YAC's |
| gel stretching | coat microscope slide with restriciton enzyme (inactive), and put the chromosomal DNA on the slide along with molten agarose. the agarose hardens, stretching the DNA..Mg2+ is added to activate the restriction enzyme, and then cut sites are largely visible under flouresecnt microscope |
| molecular combing | a different way of stretching DNA; a coverslip with silicon coating is dipped into solution of DNA, and removed slowly at a rate of .33 mm/s, producing a comb of parallel molecules |
| FISH (flourescence in situ hybridization) There also exists fiber-FISH, abandoning the use of chromosomes for purified DNA, which would resolve to 10 kb apart | like optical mapping, this technique enables the position of a marker on a chromosome to be directly visualized , but instead of a restriction site, the marker is a sequence visualized by hybridization with a flourescent probe....intact chromosomes visualized --DNA must be denatured, single stranded, so that it can hybridize with the probe ...Often, the probe contains many repeated sequences like Alu, and so unmarked DNA from the organism being studied is included in the mix, so that it may hybridize with the repeats, so that the hybridization is driven wholly by the unique sequences |
| Usedto be metaphase chrmosomes, but they are often TOO condensed, cant give a good enough picture ..now we use interphase chromosomes because they are the loosest, but then they dont allow for measurement from the tip of the short arm- called the FLpter value)..so often used after a preliminary map has been obtained | What chromosomes are used for FISH? |
| STS (sequence tagged site) mapping | simply a short DNA sequence (100-500 bp) that is easily recognizable and occurs ONLY ONCE in the chromosome or genome being studied--in order to map a set of STSs, need a collection of overlapping DNA fragements |
| 2 crtieria for an STS | 1. sequence must be known, (for PCR amplifcation purposes) 2. must be unique in the genome |
| 1. ESTs (expressed sequence tags) 2. SSLP's (simple sequence length polymorphisms) 3. random genomic sequences | 3 most common sources for STS's |
| ESTs (expressed sequence tags) | [a source for STS's]..short sequences obtained by analysis of cDNA clones (DNA made from mRNA)...assume that they are unique gene, and not from a gene family with the same or similar seuqnces |
| radiation hybrid | one possible mapping reagent for STS mapping----a rodent cell that contains fragments of chromosomes from a second organism--using X rays to irridiate the human chromosome, and subsequently fusing them with the non irradiated cells of a hamster --the hybrids are the ones that can actually survive in the HAT medium (given to cell lines that are TK and HFRT deficient), because theyve received the conferred ability from human genes to survive it. |
| mapping reagent - radiation hybrids OR library of clones | the collection of overlapping DNA fragments or chromosome to be studied (for STS mapping) |
| flow cytometry of Facs | a way of sorting out different kinds of chromosomes...a mixture of flourescently stained chromosomes passed through a very small aperture so that they flal one by one. the flourescence detector identifies the correct chromosome by the flourescence, and applies an electric charge to these drops- they are deflected into another beaker |
| HAT medium | used for the radiation hybrid hamster cells...the cell line is deficient in TK and HFRT enzymes, they would die in a medium of Hypoxanthine, Aminopetrin, and thymidine |
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