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lab practical
| Question | Answer |
|---|---|
| among the crosses below which will give a 1:1 ratio of genotypes | AAbb x aaBB |
| pairs of alleles that distribute randomly in gametes without regard to other pairs of alleles illustrate the | law of independent assortment |
| what is the probability of having an offspring with the genotype of aabbccdd if both parents were heterozygous at all loci | 1/256 |
| x - linked dominant | trait is seen in every generation and females have it more than males |
| x - linked recessive | mutation never passes from father to son even though more males have it than females |
| a purebred plant is one that is homozygous dominant at all loci | true |
| meiosis produces rearranged genes during anaphase II | false |
| the genome is our --- | hereditary code |
| what is PCR | a procedure that is used to amplify a specific sequence of your own DNA in a test tube - used to detect specific mutations that may cause genetic disease |
| molecular biology | the study of genes and the molecular details that regulate the flow of genetic information from DNA to RNA to proteins, from generation to generation |
| biotechnology uses this knowledge to | manipulate organisms DNA to help solve human problems |
| PCR has made an impact on what four main areas of genetic research | gene mapping, cloning, DNA sequencing, and detection |
| PCR is used as a tool to | detect specific mutations that may cause genetic disease, in criminal investigations, and the sequencing of the human genome |
| you make dna from which direction | 5' - 3' |
| you read dna from which direction | 3' - 5' |
| what is needed to perform PCR | primer, template, dntp, taq, buffer |
| what temperature denatures | 94 C |
| what temperature anneals | 64 C |
| what temperature does polymerization occur | 72 C |
| why is it necessary to chelate the metal ions from solution during the boiling/lysis step at 100 C? | chelating the ions neutralizes the cleavage function |
| what would happen if you didnt use a chelating agent such as the insta gene matrix | if you dont chelate the metal ions, cleavage would occur |
| what structures must be broken to release DNA from a cell | cell and nuclear membranes |
| why do you think the DNA is stored cold with the instagene matrix after boiling the samples | in the event that their are remaining enzymes it slows down the activity to prevent it from damaging the DNA |
| why is it necessary to have a primer on each side of the DNA segment to be amplified | the primers act as a starting or reference point for taq DNA |
| how did taq DNA acquire its name | it is named after thermus aquaticus |
| why are their nucleotides in the master mix | they are incorporated into dna strands |
| what are the other components and their functions | dna template - provides sequence deoxynucleotides - the raw material of the dna primer - defines amplified region mg ions - the catalyst salt buffer - gives pH for reaction |
| describe the three main steps of the PCR amplification | denature anneal extend |
| explain why the precise length target DNA sequence doesnt get amplified until the third cycle | to simplify the process, this is due to the fact that the number of DNA molecules double each time and you need a certain amount, thus three cycles |
| explain the differences between introns and exons | introns are included in the mrna that is extracted and it is non coding while exons are coding molecules that are linked together |
| why do the two possible PCR products differ in size by 300 base pairs | only one includes an insert |
| explain how agarose electrophoresis separates DNA fragments, why does a smaller DNA fragment move faster than a larger one | it separates depending on size, smaller DNA fragments are able to travel further because they are not as heavy |
| what kind of controls are run in this experiment? why are they important? could others be used? | +/+ -/- -/+ there are others that are not templated. these three act as reference points so we can determine our alu insert |
| dermatoglyphics | the study of epidermal ridges |
| finger print patterns can be classified | arches, whorls, and loops |
| finger print characteristics of trisomy 21 patients | fingers primarily ulnar loops; radial loops o fingers 4 and 5 |
| loop pattern characteristics | loop has a triradius and core |
| triradius | is a point at which three groups of ridges coming from three directions meet at angles of about 120 degrees |
| frequency of arches in general population | 5.0% |
| frequency of radial loop in general population | 5.4% |
| frequency of ulnar loop in general population | 63.5% |
| frequency of whorl in general population | 26.1% |
| average TRC for males | 145 |
| average TRC for females | 126 |
| 4 DNA nucleotide bases | Adenine Cytosine Guanine Thymine |
| DNA is composed of | sugar phosphate and bases |
| what does PCR stand for | polymerase chain reaction |
| primer | direct the polymerase to specific areas on the template DNA strand that flank the target DNA sequence |
| taq polymerase | builds the new DNA molecules from free deoxynucleotides added to the reaction |
| Denaturation | Heating to 95°C is used to break the hydrogen bonds between nitrogenous bases and separate the strands of DNA. |
| priming | After cooling, the primers attach to their complementary sites on the separated DNA strands |
| extension | The sample is warmed back up to 72°C and the polymerase begins synthesizing new strands starting at the primers |
| gel electrophoresis entails | Preparing the DNA samples Using an electrophoresis gel and chamber to separate the fragments Using a staining process to visualize the gel results after sample fragments are separated by electrophoresis |
| what is bromophenol blue used for in PCR | to visualize where the samples are located in the gel |
| what is glycerol used for in PCR | helps the sample sink to the bottom of the well and not float on top of the buffer solution. |
| how do the gel wells work | One well contains a DNA ladder made up of a collection of DNA fragments of known length. As these spread through the gel, they provide a reference point to any sample fragments that line up in the same location. |
| do smaller or larger fragments move faster | smaller, because they do not weigh as much and simply get further |
| how do you see where the DNA fragments are in the gel | the samples all contain a stain, like ethidium bromide, which binds to DNA molecules for detection and allows visualization of the DNA bands in the gel |
| what does the master mix contain | Taq polymerase, primers, deoxynucleotides, and polymerase buffer solution |
| DNA | Double helix structure containing base pairs used as the basis of genetic information |
| DNA ladder | Solution of DNA fragments of known size used to compare unknown DNA to estimate fragment size |
| electrophoresis chamber | Buffer-filled box where a separation gel is placed, and a current is applied to separate DNA fragments by size |
| SYBR green I | A safer alternative to Ethidium Bromide used to visually detect the location of DNA bands in the electrophoresis gel |
| what is used to visually detect the location of the bands in PCR | SYBR green I or ethidium bromide |
| loading dye | A solution added to an electrophoresis sample to give it color and density |
| microcenterfuge | Small tabletop centrifuge that uses centripetal force to separate substances by density |
| primer | Short DNA fragments with a known sequence |
| restriction enzyme | An enzyme that cuts DNA at specific recognition sites called restriction sites; EcoRI and Psti enzymes are examples |
| taq polymerase | A DNA polymerase enzyme adapted to working at high temperatures without denaturing |
| the wells of the agarose gel are near | the black (negative electrodes) |
| source of GFP | bioluminescent jellyfish |
| genetic transformation | change caused by genes and involves insertion of a gene into an organism in order to change the organism's trait |
| how can the gene that codes for GFP be switched on | by adding the sugar arabinose to the cells nutrient medium |
| how is selection for cells that have been transformed with pGLO DNA is accomplished by | growth on antibiotic plates |
| transformed cells will appear --- on plates not containing arabinose and --- on plates containing arabinose | white and fluorescent |
| to genetically transform an organism, you must insert the new gene into every cell in the organism. which organism is better suited for total genetic transformation, one composed of many cells, or a single cell | single cell |
| scientists often want to know if genetically transformed organism can pass its new traits on its offspring and future generations. to get this information, which would work best, one where each generation reproduces more quickly or more slowly | one where each new generation develops and reproduces more quickly |
| whats the best choice for genetic transformation: bacterium, fish, or mouse, | bacterium as they are both single celled and fast producing |
| goal of genetic transformation | to change and organisms traits |
| how could you use two LB/agar plates, some E. coli, and some ampicillin to determine how E. coli cells are affected by ampicillin | you could use two different plates with ampicillin on one plate.if ampicillin has a negative affect, fewer colonies will appear, equal amounts means it has no effect |
| what would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells | if not resistant, ampicillin would kill the bacteria on the E. coli cells |
| genetic transformation involves the insertion of | some new DNA into E. coli cells |
| plasmids | small circular pieces of DNA usually contains genes for more than one trait |
| how would you get more reporter gene | add more pheromone |
| how to purify GFP | hydrophobic interaction chromatography |
| AB represents what two phenomenons | co dominance and multiple alleles |
| what was the GFP promoter | arabinose |
| genetic engineering | process used to insert genes coding for new traits into a plasmid |
| pGLO plasmid carries | GFP and bla |
| three steps for transformation procedure | 1. use a transformation solution of CaCl2 2. carry out a procedure referred to as heat shock 3. provide them with nutrients and a short incubation period to begin expressing their newly acquired genes |
| to move pGLO plasmid DNA through the cell membrane you will | 1. use a transformation solution of CaCl2 2. carry out a procedure referred to as heat shock |
| for transformed cells grow in the presence of ampicillin you must | provide them with nutrients and a short incubation period to begin expressing their newly acquired genes |
| what are the four plates you'll have in pGLO transformation | LB/amp x 2 LB/amp/ara LB |
| how to perform heat shock | put the tubes in heat, the ice, then heat, then ice, then incubate |
| what is meant by a control plate? what purpose does it serve | a control plate is used to compare results, because they lack the plasmids, the -pGLO plates are the control group |
| 3 parts of a nucleotide | a deoxyribose sugar with a phosphate connected to one end, and one of four nitrogen-containing bases (adenine, thymine, cytosine, and guanine) on the other side of the deoxyribose sugar |
| transformation is a form of | horizontal gene transfer |
| The transformation process requires plasmid DNA to pass through the cell membrane. This is achieved by making the cells | competent, meaning they are able to allow large DNA molecules to pass through. Exposing cells to a solution of cold calcium chloride starts the process |
| what does a heat shock do | increases the permeability of the cell membrane |
| how to differentiate between transformed and non transformed cells | cells should be plated on a selective medium that only allows transformed cells to grow |
| In this simulation, you transform bacteria with a jellyfish gene whose product is | green fluorescent protein |
| how is pGLO transformation performed (GFP) | you will mix bacteria w/ the pGLO plasmid followed by a heat shock procedure to transform the cells. Broth will be added to help the cells recover after taking up the plasmid, & then the cells will be plated on a variety of selective & nonselective media |
| ori site | location for DNA polymerase to start replication of the plasmid |
| bla gene | confers resistance to ampicillin, allowing transformed cell and its progeny to survive on media containing ampicillin |
| GFP gene | taken from jellyfish, it encodes for a green fluorescent protein, the gene of interest in this simulation |
| araC gene | regulates transcription of the GFP gene, which is active in the presence of arabinose |
| LB (Luria-Bertani) agar | This is a nonselective medium so all cells will grow |
| How many cycles of PCR can run before errors start to occur? | 30 or 40 |
| DNA fingerprinting procedure summary | We added restriction enzymes to DNA samples, incubated the samples, then added dye, and loaded the agarose gels for analysis. This is also called RFLP analysis. |
| What is the purpose of electrophoresis? | separate DNA fragments by size and the negative charge on phosphate backbone |
| What was in the transformation solution? | calcium chloride |
| what were the control plates | LB plate LB/amp |
| What was the purpose of ampicillin in the transformation experiment? | to select for the transformed cells |
| How did we purify GFP? | Hydrophobic interaction chromatography which separates proteins based on their hydrophobic properties |
| rank the hydrophobic chromatography buffers from least to most salty | TE/elution -> wash -> equilibration -> binding |
| purpose of equilibration buffer in hydrophobic interaction chromatography | balances the pH and salt concentration of the column before sample loading |
| what is the UV light used for in the transformation experiment? | to view GFP |
| what is the incubator used for in the transformation experiment? | provides optimal growing conditions for bacteria |
| what is the shaking incubator used for in the transformation experiment? | aerates bacteria |
| what is the centrifuge used for in the transformation experiment? | pellets bacteria and separates it from liquid |
| what is lysozyme used for in the transformation experiment? | digests bacterial cell wall |
| what is the freezer used for in the transformation experiment? | expands cytoplasm and breaks cell wall |
| polygenic inheritance | combined effect of two or more genes on a single character |
| What materials did we use to perform PCR? | - InstaGene Matrix that traps excess metal ions/cofactors - protease also within the matrix that breaks down protein contaminants |
| Alu inserts | short, repetitive DNA sequences that are present in the human genome |
| what is the point of hardy - weinburg | we can determine if the population is in equilibrium or if there is evidence of factors such as natural selection or genetic drift affecting the gene pool |
| What are the five Hardy-Weinberg assumptions? | 1. population is large 2. random mating 3. no selection 4. no mutation 5. no migration or genetic drift |
| what does p^2 represent | individuals who are homozygous dominant |
| what is a carrier frequency | the proportion of individuals in a population who carry a recessive allele but do not express the associated trait |
| the physical properties of you or any organism are due to its complement of | proteins |
| which is controlled by its complement of | genes |
| which are located on | chromosomes |
| made of --- and --- | DNA and proteins |
| a proteins function is dependent on its --- and this is dependent on its sequence of --- | shape and amino acids |
| which is controlled by a sequence of --- via the process of --- and --- | mRNA via transcription and translation |
| a heritable change in nucleotide sequence is called a --- which if in a gene can form a variant of that gene or an --- | mutation and allele |
| some of which may code for a completely functional protein which could be called called --- or may now code for a non functional protein and be referred to as | recessive |
| null hypothesis | the statement that there is no significant difference between the observed and expected values |
| how do you calculate chi squared | This is done by taking the difference between the observed and expected values, squaring the differences, dividing each squared difference by the expected value, and adding up all the resulting values |
| degrees of freedom | the number of categories minus one |
| Restriction enzymes | are produced by bacteria to cut the DNA of viruses that infect them |
| DNA profiling has three key steps: | Prepare a restriction enzyme digest of the DNA samples Use an electrophoresis gel and chamber to separate the DNA fragments by size Use a staining process to visualize and then analyze the DNA fragments in the gel |
| DNA exists in all cells but is housed inside the --- of eukaryotic cells | nucleus |
| how is DNA released in DNA isolation | cell lysis |
| what is the point of detergent in DNA isolation | it emulsifies membrane lipids and proteins, keeping them from interacting with DNA |
| what exactly is a dihybrid cross | cross between two different traits (ex. hair and eye color) |
| phenotype | observable characteristic |
| Explain how agarose electrophoresis separates DNA fragments | - a current is ran through a gel made of agarose - DNA is mixed with loading buffer, containing tracking dye & substance to cause sample to sink - sample is pipetted in well- electrical current - DNA, which is neg. charged, moves toward pos. electrode |
| What structures must be broken to release the DNA from a cell? | the nuclear membrane, plasma membrane, and cell wall |
| What materials did we use to perform PCR? | - InstaGene Matrix that traps excess metal ions/cofactors - protease also within the matrix that breaks down protein contaminants |
| hydrophobic interaction chromatography | a technique which can be used to separate or purify proteins from other molecules |
| How did we purify the GFP from the transformation experiment? | hydrophobic chromatography |
| List 4 things that led to the change in color after the addition of the pheromone in yeast experiment | Binding of pheromone to receptor Activation of G proteins Activation of MAP kinase Activation of transcription factors |
| explain southern blotting technique | 3 step process for use in detecting specific DNA fragments: -separate DNA molecules -transfer separated DNA molecules -hybridize to radioactive specific DNA probe |
| DNA fingerprinting procedure summary | We added restriction enzymes to DNA samples, incubated the samples, then added dye, and loaded the agarose gels for analysis. |
| What is RFLP analysis? | the process of breaking down a DNA sample into various fragments using restriction enzymes, and visualizing and analyzing those fragments through the process of gel electrophoresis |
| what is the purpose of karyotyping | to examine chromosomes in a sample of cells |
| how is karyotyping performed | The chromosomes are arranged in pairs, with the pairs arranged in order by size and banding pattern. they can then be analyzed and examined to determine genetic disorders, abnormalities |
Created by:
allikennedy18