Biotechnology exam 2
What are the two approaches of studying gene expression? What do they both start with?
- two approaches: both start with RNA→cDNA -Real time or quantitative (qPCR)
What does the reaction tube require in chain termination sequencing?
-Single primer annealing to denatured DNA template - All 4 dNTPs - DNA Polymerase - Dideoxynucleotide (ddNTP) which has a 3' H instead of 3' OH on the deoxyribose so it cannot form a phosphodiester bond with the incoming nucleotide & so gets
What is a major challenge of biotechnology
A challenge of biotechnolofy is to understand and control the protein folding in the manufacturing process
What are the advantages and disadvantages of PCR?
Advantages: -Rapid and aeffective compared to using DNA libraries Disadvantages: -Need to know something about the DNA sequence that flanks the gene of interest to design primers
What laboratory technique is used to study mRNA levels in a sample when the level of detection is below that of northern blot? A. southern blot b. western blot C reverse transcription PCR D. FISH E. None
C reverse transcription PCR
What do DNA sequence primers do?
DNA sequencing primers flank ends of multiple cloning sites.
What additional enzymes studied in Chapter 3 would need to be mass produced for recombinant DNA technology?
Restriction enzymes
Why does the size of the DNA cloning vector matter?
Small enough to be separated from chromosomal DNA of host plasmid
What does a proteins structure and function depends on
The structure & function of a protein depends on protein folding
Where are RNA polymerase promoter sequences used for?
used for transcription in vitro and in vivo
What is reverse transcription PCR used for?
used to study mRNA levels when level of detection is below that of Northern
What is protein engineering
• Biotechnology is able to design and build new proteins in the lab -Invent proteins that are tailored for specific applications
What are the most common secondary protein structures?
▪ Alpha Helix: amino acids form right handed spiral as hydrogen bonds stabilize the structure linking an amino acid ▪ Beta sheet: hydrogen bonds link nitrogen and oxygen atoms forming sheets that are parallel (chains run in same direction or antiparallel (chains alternate in direction)
What are DNA libraries? What are they screened for? What are the two types? What analogy was used in class?
- Collections of cloned DNA fragments from a particular organism contained in plasmid vectors within host bacteria (ex. E.Coli) - Screened to pick out different genes of interest -Two types of Libraries: Genomic DNA Libraries and Complementary DNA Libraries(cDNA libraries) - Find one sentence in a Harry Potter book in 10 min; if each person gets one page to read its faster to find the sentence
What were the first and second recombinant human protein marketed? when? How was this done? What can this technique do?
- The first was insulin in 1982 - The second was growth hormone in 1985 -Clone human insulin DNA sequence into a plasmid and the bacteria cells were then used to synthesize the protein product of the cloned gene -Can generate lots of pure protein via this technique.
What is calcium chloride transformation of bacterial cells used for? Is it efficient? Describe the process. What does calcium chloride get rid of?
-A process for inserting foreign DNA into bacteria -Inefficient -Process: 1. Treat bacterial cells with calcium chloride, while chilled on ice for 30 minutes. (chilled to maintain membrane integrity and stabilize it.) 2. Add plasmid DNA to cells chilled on ice. 3. Heat the cell and DNA mixture (30 sec @ 42°C). 4.Plasmid DNA enters bacterial cells and is replicated and express their genes -Calcium Chloride gets rid of the net negative charge on DNA
What are the advantages of PCR
-Amplify millions of copies of target DNA from small amounts of starting material in short period of time -To calculate the number of copies of target DNA starting with 1 molecule of DNA use this equation 2^n in which N represents the number of PCR cycles
What is electroporation and what are its advantages?
-Apply brief pulse of high voltage electricity to create tiny holes in the bacterial cell wall that allow the DNA to enter -Rapid -requires fewer cells -can be used to introduce DNA into other cell types - more efficient
What is the process of colony hybridization?
-Bacteria with recombinant DNA grown on agar plate - Nylon or nitrocellulose filter is placed over the plate - Treat filter with alkaline solution to lyse the cells and denature the DNA; then bake filter or UV exposure - Denatured DNA binds to filter as single-stranded DNAo Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye. - Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization
What can real time or quantitative PCR (qPCR) do? What does it need? What does the reaction tube contain?
-Can quantify amplification reactions as they occur in real time -Need special thermal cyclers that use a laser to scan a beam of light through the top or bottom of each PCR reaction. -Each reaction tube contains EITHER a fluorescent dye containing probe with quencher, or DNA binding dye that emits fluorescent light when illuminated by the laser
What happens within bioreactors?
-Cells are stimulated to produce the target proteins through precise culture conditions that include a balance of temp., oxygen, acdity, and other variables -At appropriate time the proteins are isolate from the cultures, tested at every step of purification and formulated into pharmaceutically active products -Must comply with FDA regulations at all stages of process.
What is the chain termination sequence? who developed it and when?
-Chain termination sequencing (sanger method) -Developed by Frederick Sanger & colleagues in 1977 -Important to determine the sequence of nucleotides of the cloned gene
What does fluorescence in situ hybridization (FISH) give?
-Chromosome location of gene and copy number o Identify which chromosome contains a gene of interest
What are proteins? What do they have? What is hydrophilic and hydrophobic?
-Complex molecules built of chains of amino acids -Have specific molecular weights and Have electrical charge that causes them to interact with other molecules -Hydrophilic: water loving -Hydrophobic: water hating
What is blue-white selection? What color is the functional LacZ and which is the non-functional LacZ?
-DNA is cloned into plasmid restriction site within lacZ gene. When it is interrupted by an inserted gene, the lacZ gene cannot produce functional beta galactosidase. -Non-functional lacZ = white colony = clone = genetically identical bacterial cells each containing copies of the recombinant plasmid HAVE INSERTED GENE -With X-gal added to media in petri plate, functional lacZ = blue colony = clones = genetically identical bacterial cells each containing recircularized plasmid; no transgene.
Why is the filter in colony hybridization washed? why is it exposed to X-ray film? what is it compared to and why?
-Filter is washed to remove excess unbound probe -Filter exposed to X-Ray film or digital camera to detect fluorescent probe -Film or picture is then compared to the original agar plate to identify which colonies contained recombinant plasmid with the gene of interest.
What is an example of how glycoproteins are used as treatment of disease
-Glycoproteins can be used as a NEW way to target and destroy B lymphoma cancer cells -How does this treatment work? o Glycoprotein is combined with a nanoparticle loaded with a chemotherapy drug o By targeting cancer cells with receptors for these glycoproteins attached to nanoparticles loaded with the chemotherapeutic agent, the effective dose of the drug is increased while protecting normal tissues
What is the process of the original Sanger method?
-Had 4 separate reaction tubes and each contained the same DNA; radioactively labeled single primer; all 4 dNTP; and a small amount of one ddNTP per tube; DNA Polymerase -Over time a ddNTP will be incorporated into all the positions in the newly synthesized strands creating fragments of varying lengths that are terminated at the ddNTP -Then the fragments are separated on polyacrylamide gel -Autoradiography used to then identify radioactive fragments. -Read the gel from bottom to top as individual nucleotides -The sequence generated from the reaction is complimentary to the sequence on the template strand in the vector sequencing 200-400 nucleotides per reaction. -Becomes nontoxic when it polymerizes -1% of the nucleotides are terminators in each tube -Smaller fragments move faster
What happens if a protein is folded incorrectly
-If protein is folded incorrectly, the desired function of a protein is lost and a misfolded protein can be detrimental -Incorrectly folded proteins can lead to disease ▪ Ex. Alzheimer's, cystic fibrosis, mad cow, forms of cancer and even some heart attacks
Assume you make lots of human insulin using a bacteria expression vector. Why wouldn't using human insulin genomic DNA for this cloning project be advantageous? What do you use instead?
-Introns and exons would allow for post transcriptional modifications -Use cDNA (copy DNA) in mRNA
What is NGS(2ndGS) with post. light sequencing? what does it utilize? what are the different chips?
-Ion Torrent PGM -Utilizes release of H+ on semiconductor chip -DNA → Ions → Sequence -One sensor per well per sequencing reaction -Direct detection of natural DNA extension -Millions of sequencing reactions per chip -There are three types (when you go up in chip the wells, price, reads, and GB increases): 314 chip, 316 chip, and 318 chip.
What is the procedure for reverse transcription PCR
-Isolate mRNA and use Reverse Transcriptase to make double stranded cDNA -Use PCR to amplify region of cDNA with set of primers specific for gene of interest -Run agarose gel to separate amplified cDNA fragments -Determine expression in the tissues you're comparing. **Amount of cDNA produced in RT PCR reaction for gene of interest reflects amount of mRNA and level of gene expression.
What does library screening rarely result in and why?
-Library screening rarely results in the cloning of full-length gene -Usually get small pieces of the gene; the pieces are sequenced and scientists look for overlapping sequences - Look for start and stop codons to know when the full length of the gene is obtained.
What is the process of qPCR
-Light emitted via fluorescence correlates with amount of PCR product amplified -Light is capture by the detector which relays info. To the computer to provide readout on amount of fluorescence -Readout is plotted and analyzed to quantitate the number of PCR products produced after each cycle.
Are post-translational modifications common? how do they affect protein activity?
-More than 1—post-translational modifications occur within eukaryotic cells -This change can affect the protein's activity by: Increasing its solubility orient proteins into membranes extended the active life of a molecule in an organism.
What could cause overlapping peaks in electropheragrams? How do you avoid this? Why are they bad?
-Overlapping peaks is bad, because how do you know which is the correct nucleotide -You can get overlapping by contamination, mis priming (primers annealing in more than one area instead of just one.), amplify a gene that is part of multigene, presence of a pseudogene. -Clone PCR product to avoid overlapping peaks
What is the 3rd generation sequencing with single molecular reads?
-Oxfod Nanopore Technologies -MinIon reads single molecules -10+ Kb, error rate approximately 5% -Sensor detects changes in ionic current at nanopore
What is plasmid DNA? What is it used for? What can it do?
-Plasmid DNA: small circular pieces of DNA found primarily in bacteria -Used as Vectors: pieces of DNA that can accept carry, and replicated other pieces of DNA -Can replicate independently of chromosome
How are proteins produced? What is the disadvantage? How are proteins produced more efficiently?
-Proteins produced via microbial or mammalian cell culture -Complicated and time-consuming process -Large batches of the desired protein produced in bioreactors
What is NGS(2ndGS) with pyrosequencing? what does it utilize? What is converted?
-Roche 454 commercial system -Utilizes pyrophosphate -Chemiluminescent (light releasing) reactions -ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate. -ATP is a substrate for conversion of luciferin to oxyluciferin that generates visible light.
Why is the Sanger method advantageous?
-Sanger method using capillary electrophoresis enables greater than 600 nucleotides to be sequenced per reaction -Was very helpful for completing human genome project -Procedure: only uses 1 reaction tube instead of 4
Assume the human genome project was not completed but you wanted to clone growth hormone from humans, what sequence would you use to design PCR primers?
-Taq polymerase puts a single adenine nucleotide on the 3' end of all PCR products -T vector that has single stranded thymine on each end so can complementary base pair with the adenine in the PCR products
What are taxman probes and what do they contain?
-Tawman probes are complimentary to specific regions of target cDNA between forward and reverse primers for PCRa. -Tawman probes contain: reporter located at 5' end of probe and can emit fluorescent light when excited by the laser without the quencher which is attached to 3' end of probe
What is the northern blot technique and what is the process?
-Technique involves analyzing mRNA produced by a tissue -Basic method is similar to southern blotting o RNA is isolate from a tissue of interest o RNA is separated by gel electrophoresis and blotted onto a nylon membrane, and hybridized to a labeled DNA probe. o Exposed bands on autoradiograph show presence of mRNA for gene of interest as well as size of mRNA o Can compare and quantify amounts of mRNA present in different tissues.
What is an important consideration when identifying and cloning a gene of interest? What is Taq DNA polymerase?
-The type of DNA polymerase used is very important - Taq DNAPolymerase-isolated fromas pecies known as Thermus aquaticus that thrives in hot springs
What is antibiotic selection? what does it not select?
-Transformed cells are cultures on plates containing antibiotics to identify recombinant bacteria and non-transformed bacteria -Does NOT select for plasmid containing foreign DNA vs recircularized plasmid with no DNA insert
What are some applications of proteins in healthcare?
-Treatment of damaged corneas: Biosynthetic corneas from synthetically cross-linked recombinant human collagen produced in yeast cells can help regenerate and repair damaged eye tissues and improve vision -Screening molecules associated with disease: Identify early predictor biomarker proteins to aid in the diagnosis of specific diseases using monoclonal antibodies (Ex: prostate-specific antigen test) -Foof processing -Textiles and leather goods -Detergents -Bioremediation
In colony hybridization what does the type of probe used depends on? What is an example of a probe?
-Type of probe used depends on what is already known about the gene of interest -Ex: use mouse or rat probe to screen a human library because many genes between these species are similar
What advantages does protein modification have?
-Use of proteins in manufacturing is a time-tested technology made it possible to produce specific proteins on demand (Ex: enzymes, hormones, and antibodies) -Use of proteins in manufacturing is a time-tested technology used in beer brewing, whine making, and cheese making
What is southern blotting? When was it developed and by who?
-Used to determine gene copy number; gene mapping; gene mutation detection; PCR product confirmation - Developed by Ed Southern in 1975
What Is northern blot analysis used for?
-Used to study gene expression -Techniques involve analyzing mRNA produced by a tissue Ex: Seminal Vesicles o Kidney o Epididymis segment A o Epididymis segment B
In Sanger sequencing what does 1 line (in the C or T area) mean? What does two lines mean?
1 line- Homozygous 2 lines- Heterozygous
Assume your plasmid has an antibiotic resistance gene. Following transformation, you grow up the ells on an agar plate containing antibiotic; here are your results: -Colonies 1, 3, and 6 are white -Colonies 2, 4, and 5 are blue Which colonies (CFU's) have the inserted gene?
1, 3, and 6
How is recombinant DNA made?
1. A restriction enzyme is used to cut out the desired gene from a strand of DNA. 2. These cuts produce DNA fraGments with cohesive ends 3. Two DNA fragments join by bade pairings 4. joint fragments form linear or circular molecule 5.DNA ligase unites the two DNA fragments which produces a molecule of recombinant DNA containing human and plasmid DNA
How are genomic libraries made? What are its disadvantages?
1. Chromosomal DNA from the tissue of interest is isolated & digested with a restriction enzyme which produces many fragments that include the entire genome 2. Vector (Ex. Plasmid) is digested with same enzyme 3. DNA Ligase is used to ligate genomic DNA fragments and vector DNA (plasmids) 4. Recombinant vectors are used to transform bacteria, and theoretically each bacteria will contain a single recombinant plasmid. Disadvantages: -Introns and extrons are clones (majority of library contains non-coding pieces of DNA) -Many organisms have very large genome, so searching for gene of interest is difficult -Time consuming
What do the numbers 1-8 represent in a reverse transcriptase PCR? Read from left to right
1. DNA ladder 2. -C primers only 3. cDNA only 4. rat kidney cDNA amplified w/action primers 5. rat kidney cDNA amplified w/defensin primers 6-8. rat reproductive tissues cDNA amp. Both primer sets
What is the 3 steps of the PCR cycle
1. Denaturing - heat to 94 ° C to 96° C 2. Annealing (hybridization) - in which primers H bond with complementary bases at the opposite ends of target sequence at 52° C to 58° C. 3.Extension -(elongation) - DNA Pol copies target DNA at 70 to 75° C
What are the four levels of protein structure?
1. Primary: the sequence in which amino acids are linked together 2. Secondary: occurs when chains of amino acids fold or twist at specific points forming new shapes due to the formation of hydrogen bonds between hydrophobix amino acids 3. Tertiary: 3 dimensional polypeptides are formed when secondary structures are cross linked (DETERMINES PROTEIN FUNCTION) 4. Quaternary: unique, globular, three-dimensional complexes built of several polypeptides
How are cDNA libraries made? What are its disadvantages? What are its advantages?
1. mRNA from tissue of interest is isolated 2.Need to make double stranded DNA from mRNA: -Short linker double stranded DNA sequences, which contain restriction enzyme recognition sites, are added to the ends of the cDNA -Cut with restriction enzyme, cut vector with same enzyme, ligate fragments to create recombinant vectors -Then transform bacteria with recombinant vectors Advantages: -Collection of actively expressed genes in the cells or tissues from which the mRNA was isolated -Introns are NOT cloned - Can be created and screened to isolate genes that are primarily expressed only under certain conditions in a tissue Disadvantages: -Can be difficult to make the cDNA library if a source tissue with an abundant amount of mRNA for the gene is not available.
What is the process of polymerase chain reaction?
1.Target DNA to be amplified is added to a tube, mixed with nucleotides (dATP, dCTP, dGTP, dTTP), buffer with MgCl2, and DNA polymerase 2.Paired set of Forward and Reverse Primers are added - short single-stranded DNA oligonucleotides (18-22 nucleotides long) 3.Reaction tube is placed in an instrument called a thermocycler 4.Thermocycler will take DNA through a series of reaction called a PCR cycle 5.At the end of one cycle, the amount of DNA has doubled 6.Cycles are repeated 20-30 times.
Assume you want to do 22 PCR cycles to amplify your DNA insert, how many copies of DNA will you have at the end of your PCR?
2^22 = 4,194,304
What laboratory technique utilizes a probe with a 5' end reporter and a 3' quencher to study gene expression? A. Northern blot B. Real time PCR C. Reverse transcription PCR D. FISH E. None
B. Real time PCR
Why can't you use DNA polymerase isolated from bacteria that live at optimally 37 degrees C
Because of the elongation step; DNA polymerase copies target DNA at 70-75 degrees C
What are bioreactors?
Bioreactor: cell system that produce biological molecules
What can Protein Engineering by directed molecular evolution do
Can induce mutations randomly into genes (mutagenic PCR) and then select the bacterium with the protein product (enzyme) that has highest activity.
What laboratory technique is used to identify which chromosome contains a gene of interest when generating a karyotype? A. southern blot b. western blot C reverse transcription PCR D. FISH E. None
D. FISH
What prevents restriction enzymes produced by a bacteria from digesting its own genome? A. restriction preventase B. Medichlorians C. Acetylated histones D. Methylated DNA E. None of the above
D. Methylated DNA
Who are Pauling and Corey?
Described secondary structures alpha helix and beta sheets in 1951 -structures are fragile because hydrogen bonds are easily broken
What is the polymerase chain reaction and who developed it? when was it developed?
Developed in 1983 by Kary Mullis (1944-2019) Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time
E.coli can be genetically engineered to produce the desired protein in the form of ________
E.Coli can be genetically engineered to produce the desired protein in the form of a fusion protein -Fused bacterial protein is usually an enzyme that will bind to its substrate and can be attached to a purification column
What is an electropherogram? what do the peaks represent?
Each of these peaks represents one nucleotide.
How can genetically engineered bacteria grow
Genetically engineered bacteria can be grown in large scale fermenters(anaerobic) or bioreactors(aerobic) o Computers monitor air in bioreactors keeping oxygen levels and temperature ideal for cell growth o When phase of cell growth is highest - the bacteria promoter must be activated to stimulate foreign gene expression
What can Protein Engineering by site directed mutagenesis do
Introducing specific, predefined alterations in the amino acid sequence through a process known as site directed mutagenesis Ex:a company was able to produce a bacteria and industrial enzymes that tolerate a cyanide concentration that is normally too high for most bacteria to survive.
When polymerase integrates a nucleotide what does it release?
Polymerase integrates a nucleotide and releases hydrogen and pyrophosphate.
Why are proteins so valuable
Production of proteins is a long and painstaking process
Directed molecular evolution
a method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids towards a user-defined goal.
What is selection?
a process designed to facilitate the identification of recombinant bacteria from bacteria that contain plasmid without foreign DNA, while preventing the growth of non-transformed bacteria
What are selectable marker genes?
allow to select for transformed colonies
What is SYBR green?
binds double stranded DNA, and as more double stranded DNA is copied with each round of qPCR there are more DNA copies to bind SYBR Green which increases the amount of fluorescent light emitted
The majority of proteins synthesized by E.coli are _________________
majority of proteins synthesized naturally by E.Coli are intracellular
If you have more mRNA does the fluorescence reaction go faster or slower?
more mRNA= faster
What is a copy number?
number of plasmids per cell
What is the procedure for FISH
o Chromosomes are isolated from cells and spread out on glass microscope slide o DNA or RNA probe for gene of interest is labeled with fluorescent dye and incubated with slides o Probe will hybridize with complementary sequences on chromosomes on slide o Slide is washed and then exposed to fluorescent light o Wherever probe has bound to the chromosomes, it is illuminated to indicate the presence of the probe binding o Do karyotype to determine which chromosome shows fluorescence o To determine which chromosomes are homologues they are aligned according to their length and fluorescent patterns of their chromatids and use to create a karyotype
What is the process of southern blotting?
o Digest chromosomal DNA into small fragments with restriction enzymes o Fragments are separated by agarose gel electrophoresis o Gel is treated with alkaline solution to denature the DNA o Fragments are transferred onto a nylon or nitrocellulose filter called blotting o Filter (blot) is baked or exposed to UV light to permanently attach the DNA o Filter (blot) is incubated with a labeled probe and exposed to film by autoradiography or digital camera o Number of bands on film represents gene copy number.
What is FISH used to detect?
o FISH used to analyze genetic disorders o FISH used to determine which cells in a particular organ or tissue are expressing the particular mRNA
What are the applications of PCR?
o Making DNA probes o Studying gene expression o Detection of viral and bacterial infections o Diagnosis of genetic conditions o Detection of trace amounts of DNA from tissue found at crime scenes o Detection of DNA from fossilized tissue
What are the two major phases used in producing proteins
o Upstream processing: includes the actual expression of the protein in the cell oDown stream processing: involves purification of the protein and verification of the function; a stable means of preserving the protein is also required Note: The choices made during upstream processing can simplify downstream processing
What is glycolysation?
post-translational modification wherein carbohydrate units are added to specific locations on proteins
What is multiple cloning site (MCS)?
recognition sites for several restriction enzymes in which DNA insert is cloned into restriction enzymes in which DNA insert is cloned into
What is the origin of replication?
site for DNA replication that allowm plasmids to replicate independently from host chromosomes
What is the process of the modern Sanger method?
▪ ddNTPs are each labeled with a different fluorescent dye ▪ Samples are separated on a single-lane capillary gel that is scanned with a laser beam ▪ Laser stimulates fluorescent dye on each DNA fragment which emits a different wavelength of light for each different colored ddNTP ▪ Emitted light is collected by a detector that amplifies and feeds this info to a computer that can run multiple capillary gels at one time = 900 bp sequence ▪ Computer converts light patterns to reveal the sequence.