Key Note Mole Gene

SNP (Single nucleotide polymorphism) Primer EST (Expressed-seq uence tag) DNA marker varies a single base among individuals at any site in genome. A short synthetic oligonucleotide is used to initiate amplification of DNA in a polymerase chain reaction (PCR). Unique fragment of DNA from the coding region of a gene, produced by the reverse transcription of cellular RNA. SSR A site in a genome that has more than one common sequence in a population. (Microsatellite) SSR genotyping involves the use of simple sequence repeats (SSRs) as DNA markers. SSRs, also called microsatellites, are a type of repetitive DNA sequence ubiquitous in most plant genomes. SSRs contain repeats of a motif sequence 1-6 bp in length. Genetic map A diagram of a chromosome with distances based on recombination frequencies-centiMorgans. Isogenic lines Lines or stocks of a species that are identical throughout their genomes except for a small region of interest. Maker-assisted selection (MAS) Phylogenetic tree Indirect selection approach where a trait of interest is selected based on a marker that is linked to a trait of interest. A tree represents the extent of relatedness of various species based on comparison of homologous DNA or protein sequences. BLAST Online bioinformatic tools are used for comparing an unknown sequence to a large number of known sequences in the databases. Genetic engineering A technique isolates, moves, delivers desirable genes, then creates genetically modified organisms. Amplified fragment length polymorphism (AFLP) a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest. Allelic variation Describes the presence or number of different allele forms at a particular locus (locus or loci = place) on a chromosome (allelic variation is sometimes used more loosely to describe the overall diversity present) RFLP RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific. Random Amplified Polymorphic DNA (RAPD) PCR markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence _ a technique to make many copies of a specific DNA region in vitro _ a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter is interested in. Bioinformatics 1.Lab Text Research GenBank subset databases EST – sequences of cDNA that have been reverse-transcribed from mRNA STS – short DNA segments with a single location in the genome HTG –‘unfinished’ DNA sequences generated by the high throughput sequence centers GSS – similar in nature to ESTs, except that the sequences are genomic in origin, rather than cDNA D3. What are the important parameters to be considered for designing “good” primers and what happens if the parameters are lower or higher the optimal range? (8 points) important parameters to be considered for designing “good” primers - Primer melting temperature (Tm):52–62°C Primer length: 18-25 bp lPrimer GC content: Optimum is 45–55%, can vary between 40 and 60% - Primers should not amplify common sequences Length of desired product: is determined by the purpose of the experiment, rather than the rules for designing “good” primers. 1.​ Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation. 2.​ A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target. 3.​ Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little. 4.​ Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting. 5.​ Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains. 6.​ Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT). 7.​ Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences. 8.​ If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification. 9.​ If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer. 10.​If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.