1. The following diagram shows a portion of a long double-stranded DNA template
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1. The following diagram shows a portion of a long double-stranded DNA template molecule. The... in the middle of the template molecule represents 1000 base pairs that are omitted. To amplify a target sequence that is highlighted with bold letters in a PCR, you need two types of primers. One type of primer binds to one strand and uses it as a template. The other type of primer binds to the other strand and uses that one as a template. From the four primers listed above and below the DNA template sequence, select and clearly circle the two types of primer that can be used to PCR-amplify the bold lettered-target sequence. Primer 2 GGACTGAAACGAATGGCTS Primer 1 SACTAGCTTGACGTAGCTT5 Template DNA 3 TCGATGA 5TGATCGAACTGCATCGAA3 5'CCTGACTTTGCTTACCGA3 Primer 3 Primer 4 2. What is histidine? What is the ploidy of the mutant yeast cells that you are crossing in the complementation experiment? Are the uncrossed mutants auxotrophs or prototrophs? 3. Briefly describe how one performs pair-wise mating of yeast strains. SD media? Can yeast that are deficient in histidine biosynthesis grow on YPD media? Define the following terms: crossing over: 4, 5. homologous chromosomes: replica plating: 6. Where do you dispose of the used pipette tips and used centrifuge tubes? Continued on next pageExplanation / Answer
1) primer 2 and primer 3 can be used to pcr amplify the bold targetted DNA sequence.
primer can bind to the complementary base pair of the targetted DNA sequence to start the extention of the complementary strand or daughter strand.
2) Histidine (abbreviated as His and encoded by CAU & CAC codons)
Histidine is an alpha amino acid found commonly in many proteins. it is found as an essintial amino acid i human diet especially in infants. It is the precursor of histamine and has an essential role in purine biosynthesis. Histidine consist of an alpha protonated amino group, deprotonated carboxylic group and a positively charged imidazole side chain. at pH below 6 the imidazole ring is in protonated form and acts as acid but at pH above 6 it is in unprotonated form and acs as base. due to this nature, histidine can be used as ligands in metalloproteins and also acts as a catalytic site in certain enzymes.
4) Yeast deficient in histidine biosynthesis can grow on YPD media but not on SD media. Because SD is a minimal media without amino acids.
5) Crossing over- it is a exchange of chromosomal segment between the non-sister chromatids of homologous chromosomes. it creates an recombinant chromosome (different set of genes with that of parent chromosomes) during the pachytene stage of prophase 1 of meiosis in a process known as synapsis.
Homologous chromosomes- chromosome with same gene sequence with other. in homologous gene pair one is derived from paternal and one fom mternal origin . Homologous chromosomes consist of same genes at same loci but may have different alleles for the same gene. e.g; in garden pea the pod color for the pea is yellow on the paternal chromosome and green on the maternal chromosome.
Replica plating- it is a microbiological technique used for screening a desired phenotype. in replica plating differnt petriplates with solid selective growth medium containing antibiotics or lacking nutrients were inoculated with the same colonies of micro-organism from the the master plate (primary plate ). the technique involves the use of velvet disk for imprinting the colonies from the primary plate onto the secondary plate. the colonies can be identified in the secondary plate that cannot grow without the specific nutrient or which is antibiotic resistant thus cells with mutation in the gene is isolated.
6) used pipette tips and centrifuge tubes were disposed in a proper discarded box which can be bought from different companies.
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