Term
| What is the practical use of DNA fingerprinting? |
|
Definition
-provides evidence in paternity and criminal cases
-can provide identification with great accuracy (unlike blood typing which can only exclude a suspect) |
|
|
Term
| What are restriction enzymes? |
|
Definition
| endonucleases that catalyze the cleavage of phosphodiester bonds within both strands of DNA |
|
|
Term
| What do restriction enzymes require for activity? |
|
Definition
|
|
Term
| How are restriction enzymes named? |
|
Definition
according to the organism form which they are isolated
-first letter of the genus followed by first 2 letters of the species (italicized)
-roman numeral at end to designate different RE by same organism
EX. E. coli.=Eco RI |
|
|
Term
| What is the typical length of a DNA recognition site? |
|
Definition
|
|
Term
| What are the 2 types of restriction enzyme cleavage? |
|
Definition
-"sticky" ends-a result of staggered cuts
-"blunt" ends-a result of cuts that are directly opposite of each other |
|
|
Term
| How is the frequency of of restriction enzyme cleavage determined? |
|
Definition
| 4^N, where N=number of bases that make up the restriction enzyme recognition site |
|
|
Term
| What is agarose gel electrophoresis? |
|
Definition
| -procedure used to analyze DNA fragments generated by restriction enzymes |
|
|
Term
| Which electrode does DNA migrate toward during electrophoresis and why? |
|
Definition
| -the positive electrode (red) because it has a strong negative charge |
|
|
Term
|
Definition
| variations in the length of the restriction enzyme fragments |
|
|
Term
| What are 2 advantages of PCR over RFLP-based DNA fingerprinting analysis? |
|
Definition
-allows for DNA fingerprinting identification using much smaller amounts of DNA
-alot faster, which allows critical question to be answered quickly |
|
|
Term
| What are the main steps of Southern Blot Analysis? |
|
Definition
-depurination/denaturation
-southern blot transfer
-non-isotopic detection of DNA
-membrane shielding
-detection
-color development
-termination |
|
|
Term
| Who first described RFLPs? |
|
Definition
|
|
Term
| What does electrophoresis mean? |
|
Definition
| to carry with electricity |
|
|
Term
|
Definition
circular, non-chromosomal pieces of DNA that can replicate in and are commonly found in bacteria and simple eukaryotes like yeast
-typically carry accessory genes separate from the organism's genomic DNA (like antibiotic resistance) |
|
|
Term
| How do scientists take advantage of plasmid DNA? |
|
Definition
| to introduce genetically engineered DNA sequences into bacterial cells using transformation |
|
|
Term
| What does genetic transformation literally mean? |
|
Definition
|
|
Term
| What is genetic transformation? |
|
Definition
| -insertion of a gene into an organism in order to change the organism's traits |
|
|
Term
| How is genetic transformation used in biotechnology? |
|
Definition
-agriculture: genes coding for frost, pest, spoilage, etc are genetically transformed into plants
-bioremediation: bacteria is genetically transformed with genes enabling them to digest oil spills
-gene therapy: sick person's cells are transformed with healthy copies of the defective gene causing the gene |
|
|
Term
| What main process did we perform in the pGLO transformation experiment? |
|
Definition
| transformed bacteria with the gene that encodes GFP (Green Flourescent Protein) through the aid of a plasmid |
|
|
Term
| What is the real-life source of GFP? |
|
Definition
| the bioluminescent jellyfish, Aequorea victoria |
|
|
Term
| What genes does the pGLO plasmid encode? |
|
Definition
-GFP gene
-antibiotic ampicillin resistance |
|
|
Term
| How can the GFP gene be switched on? |
|
Definition
| presence of sugar arabinose in cells' nutrient medium |
|
|
Term
| What are the 3 main steps of transformation? |
|
Definition
-move pGLO plasmid DNA through the cell membrane by
1. using a transformation solution w/CaCl2 (calcium chloride)
2. heat shock
-allow transformed cells to grow in presence of ampicillin
3. provide w/nutrients and short incubation period |
|
|
Term
| Why should you incubate agar plates upside down? |
|
Definition
| to avoid condensation from dripping onto culture and messing it up |
|
|
Term
How many base pairs do plasmids typically contain?
How many base pairs do bacteria contain? |
|
Definition
-1,000-100,000 bp
-several million |
|
|
Term
| Why are plasmids extremely important tools in molecular cloning? |
|
Definition
| they are useful in propagating foreign genes |
|
|
Term
| What are plasmids that are used to propagate foreign genes called? |
|
Definition
|
|
Term
| What type of molecule does plasmid DNA naturally exist as? |
|
Definition
|
|
Term
| What does supercoiling in a plasmid molecule result from? |
|
Definition
| alterations in the winding of the 2 DNA strands around each other |
|
|
Term
| What is another name for supercoiled plasmid DNA? |
|
Definition
|
|
Term
| What enzymes cause supercoiling in a cell? |
|
Definition
|
|
Term
| What are the biological consequences of supercoiling? |
|
Definition
-allows DNA to fit in cell
-influences gene expression |
|
|
Term
| What is relaxed form of plasmid DNA called? |
|
Definition
|
|
Term
| What type of bonds must break in order for a supercoiled DNA to become relaxed? |
|
Definition
|
|
Term
| What can cause nicks in supercoiled DNA? |
|
Definition
-endonucleases, such as DNAse I, which randomly nick
-mechanical manipulations as well |
|
|
Term
| What are the multimers of plasmids called? |
|
Definition
| catenanes (dimer, trimer, tetramer, etc) |
|
|
Term
| What are some factors that determine the rate at which DNA moves through agarose gel? |
|
Definition
-size of DNA
-shape of DNA
-type of buffer
-gel concentration
-applied voltage |
|
|
Term
| Of the different forms of plasmid DNA, arrange them in order of fastest moving to slowest moving in agarose gel. |
|
Definition
| supercoiled>linear>nicked circles>dimer>trimer>etc. |
|
|
Term
| Why does RNase survive in the presence of cell lysis solution? (in the plasmid transformation experiment) |
|
Definition
|
|
Term
| What was the cell lysis solution used in the plasmid transformation solution and what does it do? |
|
Definition
-detergent sodium dodecylsulfate (SDS)
-dissolves cell membrane and denatures proteins |
|
|
Term
| What does the potassium in the plasmid transformation experiment do? |
|
Definition
| causes SDS and associated membrane fragments/proteins to precipitate |
|
|
Term
| What is DNA profiling and in what situations is it used? |
|
Definition
-the use of molecular genetic methods to determine the genotype of a DNA sample
-crime scenes, missing persons, mass disasters, human rights violations, and paternity |
|
|
Term
|
Definition
| produces large amounts of a specific piece of DNA from trace amounts of starting material (template) |
|
|
Term
| What is "the true power of PCR"? |
|
Definition
| the ability to amplify the precise sequence of DNA that a researcher wants to study or manipulate |
|
|
Term
| What is required for PCR? |
|
Definition
-template strand
-DNA polymerase
-2 DNA primers
-4 nucleotide building block subunits of DNA |
|
|
Term
| What are polymorphic DNA sequences in humans? |
|
Definition
-the sequences in humans that vary between different individuals
-only account for <0.5% of human genes
-are the sequences used in forensic applications |
|
|
Term
What is a locus?
What are alleles? |
|
Definition
-genetic address
-different forms of a gene |
|
|
Term
| What DNA sequences are used in forensic labs? |
|
Definition
| non-coding regions that contain segments of short tandem repeats (STRs) |
|
|
Term
| What are the 3 steps in PCR? Briefly describe each. |
|
Definition
1. denaturation: melting DS into SS at 94 degrees
2. primer annealing: complementary DNA strand hybridization via DNA primers at 52 degrees
3. extension: DNA strand synthesis via DNA polymerase at 72 degrees |
|
|
Term
| What is the thermophilic bacterium from which DNA polymerase was isolated from? |
|
Definition
| Thermus aquaticus-italicized (Taq) |
|
|
Term
| What does the master mix for PCR contain? |
|
Definition
-individual building blocks of DNA (nucleotides of dNTPs)
-special buffer
-salts
-magnesium chloride |
|
|
Term
| What are cofactors in PCR and what is their purpose? |
|
Definition
| -salts and magnesium ions (needed for Taq DNA polymerase to perform optimally) |
|
|
Term
| What is an autoradiograph? |
|
Definition
| an exposed sheet of x-ray film which is use for DNA sequencing analysis |
|
|
Term
| What are the 2 approaches to DNA sequence analysis? |
|
Definition
1. organic chemical reactions with DNA bases: tedious, labor-intensive
2. enzymatic process (dideoxy method): what we did in lab, quite fast |
|
|
Term
| Briefly describe the enzymatic process of DNA sequencing. |
|
Definition
| uses the Klenow fragment of E.coli DNA polymerase I to make a DNA copy of the region to be sequenced |
|
|
Term
| What does a specialized cloning vehicle in DNA sequence analysis do? |
|
Definition
| facilitates rapid analysis |
|
|
Term
|
Definition
| short region of DNA (about 57 base pairs) which contains several unique restriction sites in which segments of DNA to be sequenced are inserted |
|
|
Term
| About how many nucleotides does the M13 virus contain in its genome? |
|
Definition
|
|
Term
| What serves as a primer for DNA synthesis from the Klenow fragment in DNA sequence analysis? |
|
Definition
| a 17-base oligonucleotide |
|
|
Term
| Why is an oligonucleotide needed in DNA sequence analysis? |
|
Definition
| to act as a primer since DNA polymerase 1 lacks the 5'-3' exonuclease activity |
|
|
Term
| How many enzymatic reactions are performed in DNA sequence analysis? |
|
Definition
| 4, one for each nucleotide |
|
|
Term
| What occurs in DNA sequencing when a dideoxynucleotide is incorporated into the DNA? |
|
Definition
| DNA sequencing is completely terminated |
|
|
Term
| Why does the incorporation of a dideoxynucleotide cause DNA synthesis to terminate? |
|
Definition
| because dideoxynucleotide lacks a 3'-OH group on the ribose ring, making it impossible for DNA polymerase to add another nucleotide |
|
|
Term
| T/F. A nested set is complimentary to the region being sequenced. |
|
Definition
|
|
Term
| What type of gel is used for DNA sequencing analysis and why? |
|
Definition
| polyacrylamide, because it can resolve fragments that differ in size by a single nucleotide |
|
|
Term
| What property of the DNA fragments in DNA sequencing analysis makes their position detectable by dark exposure band on the sheet of x-ray film? |
|
Definition
| they are radioactively labeled with ^32P |
|
|
Term
| What is the sequence in which the nucleotides are positioned on the autoradiograph (complementary to the electrophoresis gel)? |
|
Definition
|
|
Term
| Who discovered that there are 4 different types of blood? |
|
Definition
|
|
Term
|
Definition
-another word for antigens
-found on the surface of RBCs, determine blood type |
|
|
Term
|
Definition
-another name for antibodies
-found in the plasma, a person has the antibody opposite of their blood type (blood type A, antibody B) |
|
|
Term
| At what age to antibody levels peak? |
|
Definition
|
|
Term
| What is the proposed theory for antibody production? |
|
Definition
| proposed that antibody production is initiated by minute amounts of A and B antigens that may enter the body through food, bacteria, etc. |
|
|
Term
| What are the percentages of each blood type in the general population? |
|
Definition
-O: 45%
-A: 39%
-B:12%
-AB: 4% |
|
|
Term
| Which blood type is the universal donor but has both A & B antibodies? |
|
Definition
|
|
Term
| Which blood type is the universal recipient and has neither A or B antibodies? |
|
Definition
|
|
Term
| What would you deduce if agglutination occurred in a suspension that only contained anti-A serum? |
|
Definition
|
|
Term
| What would absence of agglutination in a sample indicate? |
|
Definition
|
|
Term
| What can happen if incompatible blood types are mixed? |
|
Definition
-erythrocyte destruction
-agglutination (clumping) |
|
|
Term
| T/F Blood typing can prove that an individual is the father of a child. |
|
Definition
| FALSE, it merely indicates whether or not he possibly could be |
|
|
Term
| What is the average life span of a red blood cell? |
|
Definition
|
|
Term
| What are the only mammals have oval shaped red blood cells? |
|
Definition
| camels and their relatives |
|
|
Term
| Who reported that rabbit sera containing antibodies for the red blood cells of the Rhesus monkey would agglutinate the RBCs of 5% of Caucasians? |
|
Definition
| Landsteiner and Wiener in 1940 |
|
|
Term
| What are the six antigens that make up the Rhesus factor? |
|
Definition
|
|
Term
| Who gave the Rhesus factor its 6 letters? |
|
Definition
|
|
Term
| Which of the 6 Rhesus factors is most prevalent in Caucasians, African Americans, and Asians? |
|
Definition
|
|
Term
| There are at least how many alleles for the Rh factor? |
|
Definition
|
|
Term
| T/F. anti-Rh antibodies are normally present in the plasma. |
|
Definition
| FALSE-they can be produced upon exposure and sensitization to Rh antigens |
|
|
Term
| What is the hemolytic disease of the newborn which is a result of Rh- and Rh+ blood mixing? |
|
Definition
|
|
Term
| How would an infant with erythroblastosis fetalis be treated? |
|
Definition
| remove the infant's Rh+ blood and replace with Rh- blood from an unsensitized donor to reduce the level of anti-Rh antibodies |
|
|
Term
| Describe the appearance and size of RBCs. |
|
Definition
-circular
-biconcave disks
-5-8 micrometers
-transport oxygen and carbon dioxide |
|
|
Term
| What does the transport of oxygen and carbon dioxide in RBCs depend largely on? |
|
Definition
|
|
Term
| How does the biconcave shape benefit a RBC? |
|
Definition
| provides an increased surface area through which gases can diffuse |
|
|
Term
| What is the number of circulating RBCs closely related to? |
|
Definition
| the blood's oxygen-carrying capacity |
|
|
Term
| Describe the appearance and size of WBCs. |
|
Definition
-9-25 micrometers
-function to control various disease conditions
-can move against current bloodstream by amoeboid movement.
-normal count: 5,000 to 10,000/mm3 |
|
|
Term
| What does it mean if a person's WBC count goes above 10,000? |
|
Definition
| there is an acute infection |
|
|
Term
| What does it mean if a person's WBCs drop below 5000? |
|
Definition
| person may have measles or chicken pox |
|
|
Term
| What is a differential count? |
|
Definition
| the differing percentage of WBCs in the blood due to particular diseases, important for diagnostic purposes |
|
|
Term
| What was Frederich Griffith's contribution to genetics and in what year? |
|
Definition
-1928
-transformed nonpathogenic bacteria into pathogenic bacteria using heat killed bacteria
-birth of analytical genetic manipulation
-has led to recombinant DNA technology |
|
|
Term
| What was Mendel's contribution to genetics and in what year? |
|
Definition
1865
-described principles by which genetic traits are passed from parent to offspring |
|
|
Term
| What gene produces the resistance to ampicillin? |
|
Definition
|
|
Term
| Why do the high alkaline conditions from the sodium hydroxide not cause the plasmid to separate but causes the chromosomal DNA to? |
|
Definition
| because most of the plasmid DNA is supercoiled and because it is so intertwined and entangled, it can not be separated |
|
|
Term
| What does the concentrated gel loading solution do in the mini prep lab? |
|
Definition
| makes the sample denser than the electrophoresis buffer so that is sinks into the wells |
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
| hormone-like molecules released by a and alpha haploid cell which mediate the yeast mating ritual |
|
|
Term
|
Definition
| the process by which the diploid nucleus goes through meiosis, producing 4 haploid cells |
|
|
Term
How discovered endonucleases and in what year
? |
|
Definition
| Dr. Werner Atber and Dr. Hamilton Smith in 1968 |
|
|
Term
|
Definition
| cleavage of a molecule, in our case DNA |
|
|
Term
|
Definition
| enzymes that cut nucleic acids within a DNA molecule |
|
|
Term
| Which 5 areas of biotechnology has PCR had a tremendous effect on? |
|
Definition
-gene mapping
-cloning
-DNA sequencing
-gene detection
-DNA profiling |
|
|
