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University of Ottawa
Colin Montpetit

Crossing-over: the exchange of chromosomal between nonsisted chromatids during meiosis is the basis of genetic recombination.Recombinant: non parental, from crossing over. This exchnage generates two new allele combination.Parental gamete: a gamete whose chromosome have undergone no genetic recombination. Only two genetically different kinds of gametes are formed. Each gamete receives the alleles present on one homolog or the other, which is transmitted intact as the result of segragation..Linkage ratio: complete linkage exists between two genes because of their close proximity, and organisms heterozygous at both loci are mated, a unique F2 phenotypic ratio results, which designate the linkage ratio.Linkage groups: genes located on the same chromosome show evidence of linkage to one another. Number of linkage groups should correspond to the haploid number of chromosomes. CentiMorgan: a unit of distance between genes on chromosomes representing 1 percent crossing over between two genes. Equivalent to 1 map unit.Chiasmata: the crossed strands of nonsister chromatids seen in diplotene of the first meiotic division. Regarded as the cytological evidence for exchange of chromosomal material, or crossing over.Frequency mapping – creating a genetic map in units of cM of the distance of 2 or more genes. Recombination frequency in adjacent intervals is additive. Two-point mapping – using only 2 genes in frequency mapping. 1) cross pure lines with contrasting phenotypes 2) Cross progeny in a testcross (revels gametes formed) 3) Analyze results (determine linkage (% recombination) and map)Three-point mapping – since max recombination frequency (distance) between 2 genes we can determine is 50%, using a 3rdgene in the middle may help overcome this limitation. To determine which one is in the middle calculate recombination frequency between all 3 genes, and the 2 with the biggest frequency are furthest apart. Another way is to look at lowest RF, the one gene that changes from parental genotype is the one in the middle The phenotypes of the progeny will reveal the types and prpeprotion of gametes that were produced in the triply heterozygote individual and therefore reveal the location and frequency of crossover events between the genes and permit the calculation of the map distance between the three genes. Genetic map: A map of the locations of polymorphic markers where order and distance is determined by recombination frequency. Physical map: A map of the locations of identifiable landmarks in the genome. Highest resolution physical map of a genome is its complete DNA sequence. Physical map: distanced measured in base pairs (bps). Types of physical maps: Cytogenic (chromosome)map (distinctive banding patters observed in stained chromosomes) cDNA Map (locations of expressed DNA along the genome. Radiation Hybrid Map-(Order of DNA markers (STS) that uniquely occur in genome. Contig (order of overlapping DNA fragments spanning the genome) Restriction Map( describes the order and distance between DNA restriction enzyme sites) Sequence Map (complete DNA sequence of genome)Complete linkage: two genes that are linked on the same chromosmes whereby the distance between the genes do not allow a crossover to occur between the genes.the genes will be inherited together as a unit as they will assort together during meiosis.Genetic markers: A gene or DNA sequence having a known location on a chromosome and linked with a particular gene or trait. Genetic markers associated with certain diseases can be detected in the blood and used to determine whether an individual is at risk for developing a disease.They are inherited variations that are used to test genetic hypothesis (which can be due to mutation or alteration in the genomic loci). Ex: short DNA sequence surrounding a single base-pair change (SNP). Markers are associated with diseases, but the marker is not the gene itself. In one family the marker could be associated with thedisease but not in a different family. If parent has a condition and a marker for it, and his son has the marker but no condition, recombination occured between X and Y thus transferring the marker to the Y, without actually transferring the disease. If both parents have condition, probably associated with diff. marker cuz from diff families. Molecular markers: is a fragment of DNA that is associated with a certain location within the genome. Molecular markers are used in molecular biology and biotechnology to identify a particular sequence of DNA in a pool of unknown DNA. Ex: Polymorphic molecular markers are the primary types of markers used. 5conditions that characterize a suitable molecular marker: polymorphic, co-dominant inheritance (inheritance an allele from mom and one from dad), randomly and frequently distributed throughout the genome, easy and cheap, reproducible Co-dominant inheritance: Must receive a marker we can see from both parents, making it codominant. Has nothing to do with expression, just how we detect it. Polymorphic marker: is polymorphic site or locus, a location in the genome where at least two versions of the sequence exist in the population at frequency of at least 1%. Ex: 2N=80,000 copies. 70000 copies have AT base pair and 10000 copies have CG base pair. SNP- A variation in a single nucleotide pair in DNA, as detected during genomic analysis. Present in at least 1 percent of a population, a SNP is useful as a genetic marker. 1SNPper1000bp=3million. Can be tested using northern, souther analysis, sequenceing and RFLP. Specific SNP sequences (restriction sites) can be recognized by restriction ensymes. Then run on gel and see if 1 band or multiple.Best detected with probes (specific SNPs).Insertion/Deletion (indels): if we look at DNA sequence itself, that will have the tendecny to lengthen or shorten the DNA, that is polymorphism.Variable Number of Tandem Repeats (VNTRs) – variable number tandem repeats, short sequences of repetitive DNA. Used as (minisatelite)molecular markers (to identify individuals; or for linkage analysis using markers or for identifying genetic conditions and carriers for a codntions.. All individuals have markers for a given VNTR, but all have different length. Inherited from parents. The tandem repeats can vary between 10-100bp. Can be tested using RFLP STR: microsatellite molecular markers: : short tandem repeats found throughout genome Middle Repetitive Sequences: Short tandem repeats 2–9 base pairs long that are found within minisatellites.(di-tripentanucelotides) These sequences are used to prepare DNA profiles in forensics, paternity identification, and other applications STR loci differ in the number of repeats between individuals RFLP: restriction fragment length polymorphism: Varition in the length of DNA fragments generated by restriction endonucleases. These variations are caused by mutations that create or abolish cutting sites for restriction enzymes. RFLPs are inherited in a codominant fashion and are extremely useful as genetic markers. RFLP analysis: Restriction digests (alleles=specific digest pattern); (blotting)Presence/absence of restrtion sites generates fragments of variable lengths of DNA fragments; hybridize with probes (southern); alelles (SNPs, VNTRs, STRs, genes) have unique patters), run on electrophoresis gel, denature DNA, filter with buffer and sponge, hybridize with probes to detect specific sequences interested in. Run these on a gel under X ray to compare individuals. All individuals have markers for a given VNTR, but all have different length. When comparing RFLPs to determine who is the true parent, whichever markers the child has must have been inherited from one of the parents. If a child has a marker,and it dosent match either parent, neither of the 2 males are the fathers. Each marker comes from a parent . Hybridizatoon with alale-specific oligonocluoeides: small probes 925-30 bases) can work if candtions (salt, temp) are adjusted. Mismatches much more significant for small probes. Having probes which change in one base pair and seeing which one will mattech with the gene of interest and you will know which allele it is, probe falls of if mismatch of 1bp. DNA sequencing: refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA. Start with a template (3’ to 5). Add primer. Add a lot of nucleotides and a tiny bit of one dideoxynucleotide. Dideoxynucleotides (Hs on both 2’ and 3’ carbons), which result in termination of sequence of polymerase. End up with fragments of different lengths.Repeat 3 times with different dideoxynucleotides. Load on a gel. 5’ will be at the bottom, so we can read the sequence off it. Quicker way is to label all 4 dideoxy with fluorescent markers, add them all at the same time, put on a gel on a computer, shortest sequences will fall of first and a laser at the bottom will detect color and write up the sequence on a chromatogram. If theres overlapping, template is contaminated. Probe: probe is a single stranded DNA that is complementary to the DNA you are trying to detect. Nucleic acid blotting- locating a genomic region, gene, or other sequence of interest from a complex mixture of DNA or RNA. Blots are used to ascertain the size and sequence organization of a gene or DNA sequence of interest; or the presence/absence of alles. Uses gene or allele specific probes.Northern blotting: An analytic technique in which RNA molecules are separated by electrophoresis and transferred by capillary action to a nylon or nitrocellulose membrane. Specific RNA molecules can then be identified by hybridization to a labeled nucleic acid probe.Southern blotting: Developed by Edwin Southern, a technique in which DNA fragments produced by restriction enzyme digestion are separated by electrophoresis and transferred by capillary action to a nylon or nitrocellulose membrane. Specific DNA fragments can be identified by hybridization to a complementary radioactively labeled nucleic acid probe using the technique of autoradiography. 1)Digest human DNA sample with Ddel 2) Run gel 3) Blot to filter, hybridize with probe 4) wash off excess probe, expose film. Restriction digests: a restriction enzyme binds to DNA at a specific recognition sequence and cleaves the DNA to produce restriction fragments. Most recognition sequences are palindromic, and restriction enzymes often cleave these sequences in an offset.Genomic DNA-The full complement of DNA contained in the genome of a cell or organism.Primers: In nucleic acids, a short length of RNA or single- stranded DNA required for initiating synthesis directed by polymerases.Taq polymerase: used to distinguish between a perfect primer match and a one nucleotide mismatch at the 3' end of the primer. This enzyme is capable of tolerating extreme temperature changes and was the first thermostable polymerase used for PCR. Polymerase chain reactions: make DNA copies (without host cell) by in vitro reaction that can amplify target DNA sequences present in small quantities. Need 2 oligonucelotides primers to start (1 complemenetray to the 3 end of one strand of the DNA to be amplified and one complementary to the 3 end of the other strand), excess nucleotides (dATP, dTTP, dGTP, dCTP), taq polymerase to add the nucleotides, buffer MgCl2 and thermocycler 1)denature DNA Primer Annealing (37-65) Extension(ttaqpolymerase) (72)(94),.Thermocycler- Incubate PCR sample tubes, changes temperature to optimal temperature for denaturation, primer annealing, etc. Electrophoresis: A technique that separates a mixture of molecules by their differential migration through a stationary medium (such as a gel) under the influence of an electrical field. DNA is charged negatively. Small fragments migrate faster than bigger fragments.Fragments are lighter on the strip if there is more of it.Linkage analysis with DNA markers: polymorphism can result in another allele marker. If we detect both markers -> heterozygote. If organism is homozygous, have to make sure marker is also homozygous. Run on gel and see how many bands there are (2-hetero). Cloning- Identical molecules, cells, or organisms derived from a single ancestor by asexual or parasuxual methods, for example, a DNA segment that has been inserted into a plasmidor chromosome of a phage or bacterium and replicated to produce many copies, or an organism with a genetic composition identical to that used in its production. Plasmid: An extrac
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