Designer Genes TWO

CRISPR intro

-Clustered Regularly Interspaced Short Palindromic Repeats -innovative technology that allows geneticists to alter the genome by adding, deleting, or changing portions of the DNA sequence. -originally identified in E. coli -CRISPR method is based on a natural system used by bacteria to protect themselves from infection to viruses -some bacteria have short DNA segments identical to DNA of bacteriophage viruses, these sequences are separated by a repeated DNA sequence this the name CRISPRs

gel electrophoresis

-in order to verify the successful completion of a PCR reaction, the product must be visualized -DNA is seperated and strained using agarose gel electrophoresis in which a clear gel matrix is created with a solidifying agent called agarose -agarose acts as resistance to a substance moving through it, the larger the substance is the more resistance it experiences (slower it goes), the smaller the substance is the less resistance it experiences (the faster it goes) -DNA has an overall negative charge so we charge the gel, DNA will be attracted to the positive pole. smaller fragments of DNA will migrate closer to positive electrode than larger fragments -in order to interpret results, a reference point must be given to compare the relative sizes of PCR products to a sample of a mixture or known lengths of DNA must be run along side of the samples to give a rough estimate of the size of the DNA (known as DNA ladder)

covid vaccine p6.

-mRNA and lipid solutions are then mixed together to create lipid nanoparticles -when the lipids come into contact with the naked strands of mRNA, electric charge pulls them together in a nanosecond. the mRNA is enveloped in several layers of lipids, forming an oily, protective vaccine particle -the newly made vaccine is filtered to remove the ethanol, concentrated and filtered again to remove any impurities and is finally sterilized -hundreds of empty vials are washed and heat sterilized -machines inject 0.45ml of a concentrated vaccine solution into each vial, enough for 6 doses post dilution, vials are sealed with foil and caped with purple lids, vials of vaccine are held for 4 weeks of testing prior to being shipped

DNA replication: corrections post replication

-mismatch pair: correction after replication is complete. an enzyme (exonuclease 1) is recruited to the area containing the mismatch, it cuts out part of the sequence containing the mismatch and this allows polymerase to synthesize new sequence thereby fixing the strand -nucleotide excision repair: mechanism to remove helix-distorting DNA lesions -"thymine dimers:" covalent links between adjacent pyrimidine bases on the same strand of DNA, distort the shape of DNA and prevents the accurate copying of DNA, which causes mutations

covid vaccine p1.

-moderna and pfizer are both genetically engineered recombinant mRNA vaccines -viral mRNA is produced in large quantities in a living "factory" such as bacterium, plant, mammalian or insect cells -starts with plasmids (small, circular units of DNA that replicate within a cell independently of chromosomal DNA and is most often found in bacteria)

cell disruption

-most common method for cell disruption and lysis are lysozome treatment and heat — used for bacterial samples -the lysozome breaks down the cell wall while the heat denatures cellular components, releasing the chromosomes -treatment with a detergent (e.g. sodium dodecyl sulphate) also causes cells to lysis - this method is more lijely ysed on cell-less starting material (cell wall will resist detergent) -cells an be disrupted by mild (to avoid fragmenting) mechanical grinding or shearing — methods include: -mortar and pestle (cell grinder) done in a hypotonic solution -sonification: the use of a probe that creates ultrasonic waves that causes cells to lyse (in hypotonic solution)

DNA replication: point mutations — silent mutations

-mutations that change the nucleotide sequence without changing the amino acid sequence

plasmids: transforamtion

-now that the plasmid vector has the gene of interest insert, it will be inserted into a host organism (this will often be E. Coli) -plasmid vectors are often transformed to E. Coli by heat-shock, a method that involves a chemical treatment of E/ coli cells with a calcium chloride solution making the cells "competent" — the treatment creates an attraction to the plasmid to the cell surface, when heat is applied, pores in the cell wall expand allowing the plasmid vector to enter -the plasmid vector used to transform the E. coli cells has a selectable marker meaning that a selective pressure (antibiotic) will be applied to the cells and only those that bear the gene of the plasmid will survive this pressure -if the cell has taken up the plasmid, the antibiotic resistance gene will allow that cell to resist the presence of that specific antibiotic such as penicillin, ampicillin, or kanamycin

DNA replication: point mutations — missense mutations

-nucleotide substitutions in protein-coding regions that result in changed amino acids -may alter biological properties of the protein

all of these are a problems with CRISPR except:

-off target effects (problem) -target site selection (problem) -homologous repair (problem) -gRNA and Cas9 binding (NOT A PROBLEM)

gene manipulation 3 basic requirements:

-open cells from the sample to expose the nucleic acids -separate the nucleic acids from other cell components -recover purified nucleic acids

primers or oligos

-primers are designs to hybridize to the 3' end of each strand of the target DNA sequence -primers should be: 1. between 15-30 nucleotides in length (ensuring that only the target sequence is amplified and you minimize no specific targets 2. GC content should be between 40-60% (ensures that primer sticks strongly to the target) 3. the Tm of each primer should be within =/- 5c of each other 4. ennd (3') on a G or C nucleotide (ensuring that the primer is "anchored" to the template DNA) 5. avoid 4 or more runs of dinucleotide repeats (like ATATATAT)

PCR: annealing

-primers bind to the target DNA sequences and initiate polymerization, occuring once the temperature of the solution has been lowered — 55c for 30 sec -allows for the synthetic DNA primers to anneal to the sequence they have been engineered to bind to, sequences are going to flank the 5' and 3' ends of the gene of interest

plasmids: using PCR

-restriction sites can be added to a gene of interest allowing for insertion of the gene into multiple cloning sites of the plasmid vector -once the gene of interest has been engineered to have restriction site flanking the gene the gene can be inserted into the plasmid vector

covid vaccine p2.

-the plasmids are engineered to carry the genetic material that codes for the part of the notorious spike protein on the surface of SARS-CoV-2 -scientists use the transformation technique to transform batches of E. coli bacteria which transport the plasmids inside their cells -following transformation, E. coli is swirled into a flaks of yellow-colored growth medium which is a sterile and warm environment that encourages the bacteria to multiply

covid vaccine p4.

-the plasmids are tested for purity and are compared against previous samples to confirm that the covid gene sequence has not changed -if plasmids pass the quality checks, enzymes are added to the mixture, the enzymes cut the circular plasmids and separate the covid genes into straight segments — a process called linearization, takes two days -any remaining bacteria or plasmid fragments are filtered out leaving one-liter bottles of purified DNA -the DNA sequences are tested again, and will serve as templates for the next stage of the process

covid vaccine p5.

-the purified DNA is then mixed with the building blocks of messenger RNA -over several hours, enzymes unwind the DNA templates and transcribe them into strands of mRNA -the mixture is moved into a holding tank, then filtered to remove any unwanted DNA, enzymes or other impurities -purified mRNA is mixed with water in preparation for making the vaccine -in a separate process, oily lipids that will protect mRNA and help it enter human cells is prepared, the lipids are measured out and mixed with ethanol (which will be removed from the finished vaccine)

plasmids: restriction enzymes

-these enzymes cut DNA at very specific sequences known as restriction sites (which are *palindromes) -when restriction enzymes are cut they leave "sticky ends" which are overhangs of DNA with no complementary bases bound -restriction enzymes evolved in bacteria as a primitive defensive mechanism against viruses

what do scientists use the electrical burst for

-to use the DNA free egg cell with a somatic containing DNA -to jumpstart cell division

for what reasons are people against the golden rice project

-too expensive -not effective enough -making a profit

During the cloning procedure, what is the purpose of inserting genes into a plasmid that give it resistance to antibiotics?

Antibiotic resistance allows researchers to select for those cells that contain a recombinant plasmid.

Why do genes of interest need to be inserted into plasmids during the cloning process?

Because you want the plasmid to carry the gene of interest — that is the point of engineering a plasmid. You use plasmids to make identical copies of a piece of DNA which is inserted into a plasmid, which is introduced into a bacteria like E. coli after which bacteria carrying plasmids are selected via antibiotics which will create more plasmid DNA.

what may happen in bacteria if gRNA was transcribed, Cas9 was produced, but there was no viral DNA that matched the gRNA

Cas9 would not be able to cut

________ refers to the fact that the sequence of one DNA strand specifies the sequence of the other strand in the double helix

Complementarity

Which of the following sequences is in the correct order for one cycle of PCR?

Denature DNA; anneal primers; primer extension.

TRUE/FALSE: plasmids are part of human DNA

FALSE (bacteria)

TRUE/FALSE: western blotting requires only 1 antibody

FALSE (uses 2?)

Which class of DNA mutation results in the substitution of one amino acid for another in the protein product?

Missense

If two DNA samples are exactly the same, what would you expect to happen if you exposed both to the same mixture of specifically designed primers and then carried out the polymerase chain reaction (PCR)?

The PCR products should be exactly the same.

It is common to use ddNTPs (dideoxynucleoside triphosphates) for sequencing DNA because, when incorporated into a growing DNA polymer, the ddNTP acts as a chain terminator, allowing the researcher an opportunity to determine the last-added base to a given chain. From your knowledge of nucleotide structure and DNA synthesis, what is the most likely attribute of ddNTPs for DNA sequencing?

The ddNTPs lack a 3′ OH (hydroxyl) group.

Following transformation of a plasmid into E. coli, how would you screen for clones that contained an insert (gene of interest)?

The host organism, E. coli, are transformed to become competent via heat-shock, the plasmid vector used to transform E. coli cells has a selectable marker meaning that an antibiotic will be applied to the cells and only those that have the gene of interest will survive the pressure (antibiotic resistance)

In class, we looked at the production of the Pfizer SARs CoV-2 vaccine. This recombinant mRNA vaccine uses lipids to envelop and protect the mRNA. In addition to their protect aspect, what is the other purpose for their use?

Their other purpose is to help it enter human cells & when mRNA,and lipids are mixed together they create lipid nanoparticles

In the PCR quantification procedure (qPCR) involving molecular fluorphores, what causes the fluorochrome to fluoresce?

There is a fluorescent dye that is detected by thermal cyclers.The dye is suppressed by the quencher and after the cleavage of the fluorophore from said quencher (by the polymerase), free dye emits a fluorescence signal which is then registered by a sensor in the thermal cyclers.

What step must first be carried out before one can amplify single stranded RNA templates in RT-PCR?

They must first be converted to complementary DNAs using reverse transcriptase.

When performing gel electrophoresis, why do molecular biologists add a "ladder" made up of nucleic acids or proteins of known size?

To help determine the size of each DNA fragment after the separation has occurred.

what is the selectable marker

a gene that allows us to select for bacteria that have taken up the plasmid ex. ampicillin

DNA posses what kind of charge

a negative charge

Which of the following is a DNA nucleotide?

a phosphate group, guanine and deoxyribose

What device is used to automatically change the temperature of the PCR reaction mixture, allowing each step in the cycle to take place?

a thermal cycler

which of the following enzymes is involved in the PCR process?

a. DNA ligase - NO b. reverse transcriptase - NO c. DNA polymerase - YES d. DNA primase - NO

Cells are made competent by...

adding calcium chloride to them

you plate bacteria on a medium containing ampicillin, you notice two colonies of bacteria, these colonies must have

an ampicillin resistance gene

what is transformation

bacteria taking up foreign DNA from the surroundings

why is Bt maize supported

because it has innate pest resilience so they use fewer chemicals

separation of proteins by SDS-PAGE (p2)

once the proteins have been denatured, they can be separated based on size by gel electrophoresis (the gel is made of a matrix of acrylamide) -acrylamide not agarose — proteins are generally smaller than DNA, the pores of agarose are larger thus proteins would run too fast in an agarose gel and the resolution (separation of proteins) would be poor; acrylamide has much smaller pores so resolution of the proteins are far greater

What gives a DNA molecule its negative charge?

phosphate groups

A small, circular, double-stranded DNA molecule that is separate from the main bacterial chromosome is called a(n) ___________.

plasmid

plasmid: MCS

plasmid vectors are always engineered to have multiple cloning cites which allow for the insertion of the gene of interst

The 3' end of most eukaryotic mRNAs contains a ______, while the 5' end has a _________.

poly(A) tail, modified guanosine cap

what is the first step in protein purification?

preparation of a crude extract

Which of the following is a technique used to encourage bacteria to take up DNA from the surrounding culture medium?

pretreatment of bacteria with calcium chloride, followed by a brief exposure to heat

in a single PCR cycle consisting of 30 seconds at 95c, 1 min at 55c, and 1 min at 72c, what is happening in the step run at 55c

primers are annealing to the DNA to be amplified

heart transplant pigs

recent breakthroughs in genetic engineering and drug development led to the recent successful transplantation of pig hearts into baboons by a team in germany. this pioneering cardiac xenotransplantation -- implanting a heart from one species into another -- could help prolong and save countless human lives • for example, the immune systems of baboons and other primates (including, presumably, humans) recognize pig hearts as "foreign" and attack them, leading to organ rejection. • in response, scientists have used genetic engineering techniques to produce pigs whose organs lack certain carbohydrates that are the principal targets of the immune system. • genetic engineering has also helped solve another problem with cardiac xenotransplantation. early experiments found that incompatibility between proteins in human blood and proteins on the lining of pig blood vessels could cause blood clots. • scientists are developing and testing pigs that are engineered to carry genes responsible for producing a human version of a protein called thrombomodulin, which keeps clotting under control.

CRISPR components

repeats: short segments of DNA, 20 - 40 bases in length, palindromes (sequence of letters that read the same left to right e.g. never odd or even) all identical. spacer DNA: not identical, each segment of spacer DNA is unique, matches perfectly with sequences from viruses cas genes (CRISPR Associated): make cas proteins, in general, helicases (unwind DNA) and nucleases (cut DNA)

why do the proteins have to be denatured first

so that the shape does not influence how far the protein will move

why do plasmids have multiple cloning sites

so that we can insert foreign genes into it

would the use of gene editing to cure sickle cell disease be somatic or germline

somatic

What is the name of the process by which recombinant DNA gets into bacteria after being picked up from the surrounding medium?

transformation

Another name for protein synthesis is _________.

translation

PCR machine

uses an automated thermal cycler or PCR machine, uses repeated cycles of heating and cooling for specific amounts of time to double the amount of DNA during each cycle

how is the guide DNA made?

using transcription

what health issue does golden rice address

vitamin A deficiency

CRISPR in bacteria p1

when the virus injects its DNA into the E. coli cell, if there was no immune system, the DNA would take over the cell and uses the cells own replication system to make multiple copies of itself and eventually kill the cell. however, when the bacterium detects the presence of viral DNA, the presence of a CRISPR system will induce an "immune response". The CRISPR-Cas system relies on two main components: a guide RNA (gRNA) and CRISPRassociated (Cas) nuclease.

how can we lyse a cell

with detergent with sonification with lysozome

PCR vs natural DNA replication

• 1- DNA helicase — replaced by heat • 2- primase — replaced by synthetic DNA primers that will target gene of interest • 3- DNA polymerase — utilizes a heat-stable DNA polymerase (taq polymerase — Thermophilus aquaticus — used because it can survive repetitive cycles of boiling) from bacteria living in hot springs • 4- template single-stranded DNA — will be supplied by lab • 5- deoxyribonucleotides or (dNTPs) — four different types of DNA nucleotides (GCAT)

genome editing with CRISPR-Cas9

• once inside the nucleus, the resulting complex will lock onto a short sequence referred to as the Pan. • the Cas9 will unwind the DNA and match it to its target RNA. • if the match is complete, the Cas9 will cut the DNA. • if we want to replace a mutant gene with a healthy copy, another piece of DNA can be introduced that carries the correct sequence. • once the CRISPR system has made a cut, this template DNA can pair up with the cut ends and recombine and replace the original sequence with the new correct sequence.

diabetes — somatic intervention

• researchers are using CRISPR-Cas9 to develop a personalized treatment for genetic forms of diabetes by replacing insulin-producing cells in patients. • the risk of transplant rejection is reduced by using the patient's own cells • the disease affects nearly 30 million americans and the total cost in the united states is estimated to exceed $300 billion per year.

sickle cell disease — somatic intervention

• researchers are working on an experimental gene therapy treatment for sickle cell disease (SCD). • the treatment would consist of using CRISPR-Cas9 to modify stem cells that are isolated from a patient's blood and then later reintroduced to produce healthy levels of fetal hemoglobin. • the higher levels of fetal hemoglobin are expected to counteract pain caused by the sickle cell mutation. • approximately 100,000 americans are affected by SCD and the total cost of medical care for SCD is estimated to exceed $1.1 billion per year. • a gene knock-in: the researchers are using CRISPR-Cas9 to replace the defective beta-globin gene with a repaired version, with the goal of creating normal, adult red blood cells and curing the disorder. • isolating stem cells — the bone marrow cells that generate all the body's red blood cells — be harvested for gene editing outside the body. after these cells are removed, the remaining bone marrow is destroyed with chemotherapy to allow space for the repaired and reinfused stem cells to grow. • the patient's blood stem cells are first treated with electrical pulses that create pores in their membranes. these pores allow the CRISPR-Cas9 platform to enter the stem cells and travel to their nuclei to correct the sickle cell mutation. • based on the researchers experience with bone marrow transplants, they predict that correcting 20% of the genes should be sufficient to outcompete the native sickle cells and have a strong clinical benefit

duchenne muscular dystrophy — germline intervention

• researchers have demonstrated the ability to use CRISPRCas9 to make genetic repairs in cells that allows them to produce dystrophin. • dystrophin is a protein that patients with Duchenne Muscular Dystrophy (DMD), a genetic disorder, cannot produce. • it's caused by frame-shift mutations in DMD, the gene encoding the protein dystrophin. • thousands of mutations have been found in DMD patients, suggesting that individually tailored gene-editing strategies may be required to repair the specific mutation present in each patient. however, because most of the mutations involve deletions clustered into a mutation hot spot between exons 45 and 55 (which terminate the dystrophin open reading frame and dystrophin protein expression in ~13% of DMD patients), skipping or reframing exon 51 to restore dystrophin expression may be a solution for many of these patient

protein purification: prepare a crude extract

• the first step in purifying intracellular (inside the cell) proteins is the preparation of a crude extract. • the extract will contain a complex mixture of all the proteins from the cell and some additional macromolecules. • this crude extract may be used for some applications in biotechnology. however, if purity is an issue, subsequent purification steps must be followed. crude protein extracts are prepared by the removal of cellular debris generated by cell lysis, which is achieved using chemicals and / or enzymes.

quantitative PCR

(qPCR) -adds two elements to the standard PCR process: fluorescent dye, fluorometer -thermal cyclers meant for use with qPCR include a fluorometer to detect that fluorescence -the fluorometer detects fluorescence in real time as the thermal cycler runs, giving readings throughout the amplification process of the PCR -is also called real-time PCR

PCR employs a heat-stable DNA polymerase called ________, originally isolated from a bacterium that lives in hot springs

Taq polymerase

restriction enzymes

cut DNA at specific sequences

molecular cloning

cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism

examples of damaging and in some cases even life-threatening conditions that are caused by something as simple as a single base letter change from an A to a T

cystic fibrosis, hemophilia, and sickle cell disease

The backbone of a DNA molecule is _________.

made up of alternating phosphate and sugar groups

what is PCR

mimics DNA replication by cells, carried out in controlled conditions in a lab

SDS-PAGE separates proteins by

molecular weight

which method is used to amplify the spike protein gene in the covid vaccine

multiplying plasmids in bacteria

DNA replication: polymerase III

(DNA polymerase III proofreads DNA before it is used to make proteins) 2 domains: -polymerase domain (5'-3') -exonuclease domain (3'-5') -> exonuclease activity: the ability to remove nucleotides one at a time from each end of a chain (exo=from the exterior end)

reverse transcriptase PCR

(RT-PCR) -modification of standard PCR technique that can be used to amplify RNA -the RNA template is first converted into complementary DNA (cDNA) via the viral enzyme reverse transcriptase -cDNA is then used as a template for exponential amplification using PCR

PCR: extension

-PCR reaction sill be heated to 72c for a time that is dependent on size of PCR product being produced: 1 min per 1,000 bp -the DNA polymerase — taq polymerase — is activated at this temp., the polymerase will now start polymerizing a new copy of the gene of interest -once extension is complete, PCR will cycle back to denaturing step and the process will repeat -the result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand -cyle is repeated many times (20-30) as most processes using PCR need large quantities of DNA, it only takes 2-3 hours to get a billion or so copies

separation of proteins by SDS-PAGE (p3)

-SDS-PAGE has two gels: -stacking gel: a lower concentration acrylamide (3-4%) gel that allows the sample in the well to form a very tight band -resolving (separating gel): has a higher acrylamide concentration than the stacking (ranging from 5-15%) and is used to separate the proteins based on molecular weight

electrophoretic transfer

-SDS-PAGE is an excellent platform for separating proteins but what if you want to see a particular protein? SDS-PAGE by itself cannot always do that -to identify particular proteins of interest, SDS-PAGE is often followed by a technique known as a western blot — this technique uses antibodies specific to your protein to identify their presence or absence in a gel (known as immunoblotting) -this technique starts with the transfer of the proteins from the gel to a membrane. this allows for the antibodies to probe for the protein as they will be on the surface of the membrane as opposed to buried within a gel

DNA replication: point mutations — frameshift mutations

-a nucleotide insertion or deletion that is not an exact multiple of three nucleotides, resulting in a shift in the reading frame of the resulting mRNA, usually leads to production of a nonfunctional protein -insertion: if you insert a single extra base, the nucleotide chain will still be read three nucleotides at a time but everything will become meaningless -deletion: if you delete a single base, once again it will become meaningless

DNA replication: point mutations — nonsense mutations

-a nucleotide substitution that creates a new stop codon -causes premature chain termination during protein synthesis -nearly always a nonfunctional product

cloning step two: plasmid vectors

-after amplification and verification via gel electrophoresis, we put gene in plasmid vector -are circular pieces of DNA found naturally in bacteria -carry antibiotics resistance genes, genes for receptors, toxins, or other proteins -replicate separately from the genome of the organism -can be engineered to be useful cloning vectors

why do we use acrylamide instead of agarose?

-because proteins are smaller than DNA -because acrylamide contains smaller pores than agarose

plasmid vector features

-can be designed with a variety of features: • Antibiotic resistance • Colorimetric "markers" • Strong or weak promoters for driving expression of a protein -resistance markers are most commonly against antibiotics — referred to as selectable marker (allows us to identify which cells have taken up the plasmid vector)

sanger method

-developed off polymerase chain reaction tech -variation of the normal PCR, instead of just using deoxynucleotide triphosphates (dNTPs), there are 4 separate reactions are run with the addition: ddATP, or ddTTP, or ddCTP, or ddGTP - the purpose of the addition of ddNTP is that the growth of the daughter strand will stop when a ddNTP is added (originally these ddNTPs where radioactively labeled — method has since been modified, now uses fluorescence) -og the sequencing reaction was run through a "slab" of gel now it is run in a tube gel

protein purification

-each protein purification step usually results in some degree of product loss. Therefore, an ideal protein purification strategy is one in which the highest level of purification is reached in the fewest steps -the selection of which steps to use is dependent on the size, charge, solubility and other properties of the target protein.

ELISA

-enzyme-linked immunosorbent assay is an immunological assay commonly used to measure antibodies, antigens, and proteins in biological samples -some examples include: diagnosis of HIV infection, pregnancy tests, detection of covid antibodies -ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured in a single experiment (plates are special absorbent plates which ensure the antibody or antigen sticks to the surface)

cloning first step: PCR

-first step of cloning is amplification of a desired gene (gene of interest) -a in vitro technique utilizing a DNA polymerase from a hyperthermophiles is used (hyperthermophiles DNA polymerase is used because it can survive repeated cycles of boiling)

DNA replication: proofreading

-happens as the new DNA strand is being made. -DNA polymerase III checks that the newly added base has paired correctly with the base in the template strand before adding the next base.

genetically cloned pets

-scientists have known that mammal cloning was feasible since 1996 • the process of cloning begins with cultured cells. • next, scientists extract unfertilized eggs from another, unrelated dog, removing them from its fallopian tubes. that animal generally isn't harmed, though the procedure is invasive. • the eggs are removed and brought to a laboratory where their nucleus is manually removed using a fine needle • this process strips the eggs of the genetic material that they contain, making the egg cell essentially a blank slate for scientists to fill with DNA of their choosing. • scientists can also achieve a similar effect with a targeted blast of ultraviolet light, which destroys the genetic material. • scientists then take one of the cultured somatic cells from the animal that they're seeking to clone and carefully insert it into the egg with a needle. • they hit the composite egg with an electric burst that "fuses" the two together. • using that method, the nucleus from the donor cell will become part of the egg and the nucleus from the donor cell will behave like the nucleus of the egg. • there's one critical difference. unlike an unfertilized egg, which has half of the necessary genetic information to make a new life—the other half is in the sperm cell—you already have a full set of genetic information, just as you would in a viable embryo. • the electrical burst also jumpstarts cell division. after a few days, assuming that the process successfully takes hold, the lab can then surgically implant the cells into yet another animal: a surrogate dog mother. treated with hormones, these surrogates can, under ideal circumstances, carry the pregnancies to term.

DNA replication: point mutations

-simplest type of mutation is a nucleotide substitution, mutations that alter a single nucleotide are called point mutations

separation of proteins by SDS-PAGE (p1)

-sodium dodecyl sulfate polyacrylamide gel electrophoresis — SDS-PAGE -most powerful technique for resolving protein mixtures, based on their molecular weight - similar to how agarose gel electrophoresis works for DNA -the different levels of protein structure affect how a protein will migrate through the gel, the structure of the proteins must be denatured such that only the size of the protein affects how the protein migrates through the gel (and not its higher-level three dimensional structures). this is accomplished using a series of chemicals

RT-qPCR: covid

-some viruses such as covid only contain RNA, in order for a virus like covid to be detected early in the body, scientists need to convert the RNA to DNA and quantify it via reverse transcriptase quantification PCR 1. sample is collected from part where covid gathers in body (throat/nose) 2. sample is treated with chemical solutions that remove proteins and fats & the RNA present in the sample is purified 3. this purified RNA is a mix of the person's own genetic material and, if present, the virus's RNA 4. RNA is reverse transcribed to DNA using enzyme reverse transcriptase, the individuals RNA sample serves as the template strand for the PCR 5. cDNA is converted to double-stranded DNA via PCR amplification & the amplification of this DNA sample is performed until the viral cDNA is detected by a fluorescent signal 6. a susbtance marked w a fluorophore is added to the PCR mixture containing a DNA template, specific primers, deoxyribonucleotides, & a thermostable DNA polymerase in a suitable buffer solution 7. the fluorescence of the dye is suppressed by the quencher, after the cleavage of the fluorophore from a quencher by the polymerase, free dye emits fluorescence signal which is registered by a corresponding sensor in a thermal cycler. (threshold: dye molecules that are not bound to double stranded DNA emit very low background fluorescence)

polymerase chain reaction (PCR)

-technique used to "amplify" small amounts of DNA -"molecular photocopying" — fast and inexpensive -important because significant amounts of a sample of DNA are often necessary for molecular and genetic analyses and studies of isolated pieces of DNA are nearly impossible without PCR amplification -invented by Kary B. Mullis, 1985

covid vaccine p3.

-the bacteria are allowed to grow overnight and are then moved into a large fermenter that contains up to 300 liters of a nutrient broth -the bacterial broth spends four days in the fermenter, multiplying every 20 minutes and making trillions of copies of the DNA plasmids -when the fermentation is complete, chemicals are added to break open the bacteria and release the plasmids from their enclosed cells -the mixture is then purified to remove the bacteria and leave only the plasmids

CRISPR in bacteria p4

-the gRNA and Cas9 come together to form the CRISPR-Cas9 system -when the matching sequence, known as a guide RNA finds its target within the viral genome, the Cas-9 cuts the DNA disabling the virus.

sandwich ELISA

-two sets of antibodies are used to detect the specific product 1. coat the ELISA plate with capture antibody, any excess unbound antibody is then washed from the plate, the capture antibody is an antibody raised against the antigen of interest 2. the sample is added, any antigen found in the sample will bind to the capture antibody already coating the plate 3. detection antibody is added, this antibody is labelled with an enzyme, they bind to any target antigen already bound to the plate 4. a substrate is added to the plate. ELISA assays are usually chromogenic, using a reaction that converts the substrate into a colored product which can be measured

PCR: denaturing

-two strands in DNA double helix need to be separated, this happens by raising the temp. of the mixture — 95c for 30sec — causing the hydrogen bonds between the complementary DNA strands to break, resulting in single stranded DNA

DNA sequencing

-used not only to determine the sequence of nucleotides in a gene but also to determine where genes are in genome -sanger method: chain-termination procedure maxam and giblert: chemical cleavage method (its use of radioactive material and the improvement of the sanger method caused it to be unsuccessful)

electrophoretic transfer p2

-utilizing the same general principle as gel electrophoresis where a current is applied and the components migrate toward the + electrode -electrophoretic transfer utilizes this to allow protein/DNA to migrate sideways out of the gel and into a membrane -once the proteins are transferred to the membrane the membrane is probed using antibodies — but antibodies are colorless -the issue is solved: antibody used in conjunction with a visual maker such as a fluorescence marker (like GFP) or an enzyme that makes a color (chromogenic) or a light (chemiluminescence)

DNA replication: mutations

-when a mismatched base isn't fixed, it is referred to as a mutation, mutations result in changes in the proteins that are made, this can be a good or bad thing - spontaneous mutations: result of natural processes in cells (during DNA replication if errors are made and not corrected in time -induced mutations: result of interaction of DNA with an outside agent that causes DNA damage (exposure to environmental factors such as smoking, sunlight, and radiation) -sickle cell anemia: caused by a mutation (GAG to GTG) in a gene that instructs the building of a protein called hemoglobin, causes red blood cells to become an abnormal, rigid, sickle shape

DNA replication: proofreading in action

-when a mismatched nucleotide is incorporated, DNA synthesis stalls due to the unfavorable base-pairing chemistry, the delay allows the mismatched nucleotide to disconnect and be replaced by the correct nucleotide. -DNA polymerase III senses that the nucleotide is incorrect, it moves the 3' end of the growing strand to the polymerases exonuclease domain. the incorrect base is removed and the strand is shifted back to the polymerase domain where it continues adding bases and extending the new strand

The nitrogenous base attaches to the ____-carbon of the nucleotide's sugar.

1'

PCR steps

1. denaturing 2. annealing 3. extension

what is the ideal PCR primer length

15-30 nucleotides

what is the minimum number of animals required for cloning on animals

3

RNA polymerase II promoters are located on the ____ side of each gene

5'

A tRNA has an anticodon with the sequence 3'-UAC-5'. What would be the sequence of the complementary codon?

5'-AUG-3'

The smallest fragment at the end of a DNA sequencing gel represents the

5′ end of the newly synthesized DNA.

If there were one tRNA for every codon that codes for an amino acid, how many different tRNAs should there be?

61

what temperature does extension happen at

72c

The initiator/start tRNA enters the ribosome at the ____ during protein synthesis.

A site

The polymerase chain reaction does not require

DNA ligase

Describe the process of cloning a DNA fragment into the multiple cloning site of the plasmid pUC18.

Plasmid and GOI are digested using restriction enzymes. GOI is cloned into plasmid using DNA ligase through the process of ligation.

In a single PCR cycle consisting of 30 seconds at 95C, 1 min at 55C, and 1 min at 72C, what is happening in the step run at 55C?

Primers are annealing to the DNA to be amplified.

what is the main difference between PCR and RT-PCR?

RNA template is used and is converted to cDNA by reverse transcriptase prior to the initiation of the PCR cycles

In an experiment, you purify a cysteine tRNA and chemically alter the amino acid attached to it converting it to alanine. You place this modified tRNA in a cell-free protein-synthesizing system with all of the other tRNAs, mRNA and other substances needed for the synthesis of proteins. What, if anything, will be different from the normal protein coded for by the mRNA in the mixture?

Since the anticodon was not altered on the modified tRNA converting to alanine, and the solution now contains 2 tRNA that contain the same anticodon but different amino acids, alanine's will randomly be added to the polypeptide chain in place of a cysteine.

TRUE/FALSE: a silent mutation is a nucleotide change, but the protein remains unaffected

TRUE

TRUE/FALSE: if we want to replace a gene with another one, we also need a piece of DNA that carries the other gene

TRUE

what is the first step of PCR

denaturing

BT Maize opposition

detractors / opposition to Bt maize suggest several challenges: • the potential for effects on non-target organisms. • gene flow between Bt maize and non-Bt maize. • whether the use of Bt maize is compatible with the other pest control methods. The potential for effects on non-target organisms • there have been no surprising effects on non-target organisms observed with Bt maize, which confirms the specificity of the Bt proteins. • most studies suggest Bt maize has little if any impact on predators and when compared with maize treated with chemical insecticides, Bt maize often results in increased biodiversity [for general reviews see (O'Callaghan et al. 2005)]. gene flow between Bt maize and non-Bt maize. • the transfer of genetic material between populations (i.e., gene flow) is often considered to be a potential problem between GE crops and their wild relatives. • in most areas of the world producing GE maize, however, production is isolated from related species that could hybridize with maize. therefore, this environmental concern is restricted to areas where wild relatives of maize occur (e.g., Mexico). • maize is an open-pollinated crop, but the large size of pollen grains limits its movement. nonetheless, some growers, particularly organic growers, demand little or no contamination from GE pollen or seed and generally object to production of any GE maize. this has been a particularly controversial issue in europe. whether the use of Bt maize is compatible with the other pest control methods. • although Bt maize is an important tool for growers, it cannot completely replace other pest control tactics. • insecticides, for example, may be necessary to control secondary insect pests. • finally, Bt maize should be especially compatible with biological control because reduced use of insecticides should lead to an increase in beneficial insects. • in general, traditional pest management practices must be maintained in order to avoid reliance on a single tactic.

traditional Sanger style DNA sequencing relies on a method called chain termination. what type of molecule is used to terminate DNA chains to create molecules of all possible lengths covering the fragment to be sequenced?

dideoxynucleotides

polymerase III notices it put a wrong nucleotide, which domain removes it?

exonuclease domain

what component allows us to quantify using PCR

fluorescent dye and fluorometer

what kind of mutation is a deletion

frameshift

what is the main difference between somatic and germline editing

germline editing is passed on to offspring, somatic is not

which DNA replication component is not used in PCR

helicase

somatic mutation

is a genetic alteration acquired by a cell that can be passed to the progeny of the mutated cell in the course of cell division. The mutation affects all cells descended from the mutated cell. However, somatic mutation differs from germline mutation in that germline mutations are inherited, while somatic mutations are limited to the individual

germline mutation

is an alteration in the genetic constitution of the reproductive cells, occurring in the cell divisions that result in sperm and eggs. Germinal mutations may affect a single gene or an entire chromosome. A germinal mutation will affect the progeny of the individual and subsequent generations of that progeny

what does RT-PCR do

it amplifies mRNA

what is the function of reverse transcriptase

it makes DNA from RNA

once a plasmid vector is sustained in transformed cell

it must be able to be replicated -the origin of replication (ori) allows for the plasmid vector to replicate — certain origins allow for thousands of copies of the vector per cell, others allow for only a few to one copy per cell

how does sanger sequencing tell us which nucleotide is where

it uses a gel to separate the product fragments by length and base pair

what accounts for the inability of dideoxynucleotide triphosphates to further polymerize in sanger DNA sequencing?

lack of hydroxyl group on the 3' carbon

what is the first step in DNA extraction

lysing the cells

germline vs somatic

somatic modifications: -somatic therapies target genes in specific types of cells in an individual: lung cells, skin cells, blood cells, retina etc. -non-inheritable and only affects the treated individual -first somatic traits occurred approx. two and a half decades ago -these mutations only show their effects in the cells where they occur germline modifications: -germline modification is applied to embryos, sperm, or eggs, and alters the genes in all the resultant person's cells -passed onto future generations -human germline editing of early embryos for research purposes began in 2015 -in most cases, germline mutations are 'silent' in the parent organism in which they originally occurred, except in cases when they affect the gamete production

CRISPR in bacteria p3

the CRISPR-associated protein is a non-specific endonuclease. it is directed to the specific DNA locus by a gRNA, where it makes a double-strand break. there are several versions of Cas nucleases isolated from different bacteria. the most commonly used one is the Cas9 nuclease from Streptococcus pyogenes.

In recombinant DNA technology, the term competency refers to

the ability of cells to take up foreign DNA

a silent mutation is one in which

the altered codon codes for the same amino acid

remove debris from the extract

the debris is removed by centrifugation, and the supernatant (the liquid above a solid residue) is recovered. crude preparations of extracellular (outside the cell) proteins may be obtained by simply removing the cells by centrifugation.

which antibody used in ELISA is bound to an enzyme

the detection antibody

a nonsense mutation is one in which

the mutant protein is shorter than normal

which similarity between SDS-PAGE and DNA electrophoresis is true?

the prepared macromolecules will both move toward the positive end

what is a nonsense mutation

the protein is truncated

what is true about spacers and repeats

the repeats separate the spacers

the rate of migration of DNA within a gel in the gel electrophoresis technique is primarily based on

the size of the DNA fragments

which gene is put into the plasmid of the pfizer vaccine

the spike protein

genome editing with CRISPR-Cas9 fun facts

the technology's many potential applications include • correcting genetic mutations • treating existing diseases in animals and humans • enhancing varieties of crops. certain diseases appear to be suitable for treatment by gene editing of some of the body's non-reproductive cells (somatic editing), while other genetic diseases might best be treated by gene editing of the reproductive cells or early embryos (germline editing)

in affinity chromatography which protein is eluted first

the unwanted proteins

what differentiates sanger sequencing from PCR

the usage of dideoxynucleotides

what do cry toxins do

they bind to midgut cells and kill the insect

Dideoxy DNA sequencing is based on chain termination with dideoxynucleotides. Why are normal deoxynucleotides also included in the reaction?

to allow production of a range of synthesis products of different lengths.

what is RT-PCR used for?

to amplify RNA found in viruses

what do we have to consider when making pigs that contain hearts for organ transplants

to get rid of the carbohydrates that cause the immune system to attack it

when performing gel electrophoresis, why do molecular biologists add a "ladder" made up of nucleic acids of known size?

to help determine the size of each nucleic acid fragment after the gel has run out

to which side with the DNA migrate in an electrophoresis gel

toward the positive side

aspects that affect CRISPR's efficiency and specificity

• CRISPR-Cas9 gene editing typically relies on the Cas9 enzyme to cut DNA at a particular target site. • the cell then attempts to repair this break using a cell's own DNA repair mechanisms (homologous repair). • however, this cell repair mechanism is not always efficient, and sometimes segments of DNA will be deleted, rearranged, or DNA bases from elsewhere will become incorporated into the gene. • researchers are experimenting with ways to increase repair efficiency, and some versions of Cas9 just bind to DNA without cutting it while a single base is changed. • CRISPR can also be used to generate small deletions to knock out a gene's function, however researchers have found that occasionally larger than expected deletions occur. no experiments yet reported have had error free results. • while CRISPR is very efficient at disabling genes, there are still technical issues associated with the repair or replacement of defective ones to be resolved. due to the possibility of off-target effects (unwanted edits in the wrong place) and mosaicism (when some cells carry the edit but others do not) safety is a major concern. • while enormous improvements continue to be made with CRISPR, it must be kept in mind that there are still significant technical hurdles to be overcome before germline editing is safe enough to be used in clinical settings irrespective of the ethical and social issues yet to be resolved.

CRISPR in bacteria p2

• Cas genes are going to transcribe and translate protein known as a CRISPR-associated (Cas) nuclease (AKA Cas9) • also going to transcribe the specific spacer DNA to make Guide RNA (gRNA) or CRISPR RNA (crRNA). • the guide RNA is a specific RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing. The gRNA is made up of two parts: CRISPR RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, which is transcribed from the spacer and TRACR RNA, which serves as a binding scaffold for the Cas nuclease.

problems with CRISPR

• accurate target site selection • guide RNA design • off-target effects • homology-directed repair • method of delivery

ethical concerns

• autonomy — derives from the ethical principle of human dignity and freedom. it follows from this principle that patients should not be treated without their informed consent or the informed consent of those such as parents • beneficence — requires that what is proposed should result in a positive outcome or benefit • non maleficence — requires an obligation to avoid bad outcomes or harm. essentially this means that there must be a balancing of the benefits, costs, and risks of any action with an attempt to maximize benefits and minimize harm • justice — relates to whether individuals have a right to a fair minimum level of health care and whether resources are allocated fairly

what is BT Maize

• bacillus thuringiensis is a species of bacteria that produces proteins that are toxic to certain insects. • it has been used as a safe microbial insecticide for over 50 years to control pest caterpillars. • Bt insecticides are popular with organic farmers because they are considered "natural insecticides" and they differ from most conventional insecticides because they are toxic to only a small range of related insects. • this is because specific pH levels, enzymes, and midgut receptors are required to activate and bind a given Cry toxin to midgut cells, which leads to pore formation in the insect's intestine and death. a "lock and key" analogy is useful to explain this specificity. If the midgut receptor is considered the "lock" and the Cry protein is the "key" then insect death only occurs when the "lock and key" match. • maize can be genetically engineered to produce these Cry toxins.

golden rice supporters

• do not share the opposition's skepticism as to whether golden rice would be an effective solution. Even if it didn't totally eliminate VAD in developing countries, it might make an impact. • planting and consuming golden rice alongside other interventions (like UNICEF's supplement program) will make more of a difference than any one intervention alone. we should use all tools at our disposal to prevent disease and lifelong disability. • supporters of the project also reject concerns over the fact that the Golden Rice Project has partners in the biotech industry and for-profit companies. The Golden Rice Project has freedom to operate under humanitarian use, therefore the technology can be provided free of charge in developing countries. • the Golden Rice Project also claims to be a sustainable project in contrast to the ongoing supplementation and fortification programs. the existing programs need millions of dollars per country and per year to run while in contrast, the Golden Rice Project supplies free technology transfer to support the developing countries.

why is BT Maize popular

• growers are attracted to the convenience of Bt maize hybrids because they allow for "in the bag" insect protection. • GE maize seed comes from the seller with innate pest resistance. functionally, this means that growers will be handling and applying fewer chemical insecticides, which has both health benefits for the growers and important environmental benefits. • it also of course means farmers can spend less time applying insecticides but still be confident in the protection of their crop from key pests. • growers are attracted to the yield protection and improved grain quality commonly found with Bt maize. • recent research suggests there has been an areawide suppression of european corn borer populations. this is beneficial to both Bt and non-Bt maize growers.

BT Maize

• growers of maize (corn), are challenged with a number of pests, but the most important are lepidopteran larvae (i.e., caterpillars) that are stalk borers, ear or leaf feeders, and coleopteran larvae (i.e., beetle grubs) that feed on roots. • the european corn borer, for example, was nicknamed the "billion dollar bug" because it cost growers over a billion dollars annually in insecticides and lost crop yields. • most maize growers rely on traditional crop protection practices to manage these insects, including biological or chemical (insecticide) methods or a balance of these methods that aims to minimize environmental impact. • however, in 1996 USA growers were introduced to commercial maize that was genetically engineered (GE) with resistance to European corn borer and other lepidopteran maize pests. • in 2003 another GE maize was introduced that killed corn rootworm larvae (beetle grubs), especially larvae of the western corn rootworm, another "billion dollar bug". • these GE plants produce crystal (Cry) proteins or toxins derived from the soil bacterium, Bacillus thuringiensis (Bt), hence the common name "Bt maize". • Bt maize has revolutionized pest control in a number of countries, but there still are questions about its use and impact.

golden rice

• the golden rice project was first introduced in 1999, when two professors Ingo Potrykus and Peter Beyer, proposed their project to Rockefeller Foundation to genetically engineer rice to increase its nutrients. • golden rice is a genetically modified, biofortified crop. • biofortification increases the nutritional value in crops. • golden rice is genetically modified in order to produce beta carotene, which is not normally produced in rice. • beta carotene is converted into Vitamin A when metabolized by the human body. we need Vitamin A for healthier skin, immune systems, and vision • rockefeller foundation supported the professor's goal to provide a sustainable biofortification approach to combat vitamin A deficiencies in developing countries. • vitamin A deficiency (VAD) is prevalent in developing countries whose diets are dependent on rice or other micronutrient-poor carbohydrate foods, which do not contain vitamin A. • the WHO estimates that about 250 million preschool children are affected by VAD and about 2.7 million children die because of the deficiency. • VAD can have numerous negative health effects such as dryness of the eye that can lead to blindness if untreated; reduced immune system response, and an increase in the severity and mortality risk of infections. VAD is one of the main causes of preventable blindness of young children from developing countries. • the golden rice project began as a proposed solution for VAD; however, despite the establishment of a Humanitarian Board and abiding by national and international regulations governing GMOs, opposition to the project has blocked the roll-out of the Golden Rice Project in developing countries.

leber disease - somatic intervention

• the world's first in vivo CRISPR study was announced in a July 2019 press release (in vivo means cells don't have to be removed, treated and re-introduced to a patient). • it aims to treat people born with a form of inherited blindness resulting from a point mutation in a gene called CEP290. the treatment involves injections directly into the retina and targets the most common cause of inherited childhood blindness. • the groundbreaking procedure involved injecting the microscopic gene-editing tool into the eye of a patient blinded by a rare genetic disorder, in hopes of enabling the volunteer to see. • the first stage of the study, which treated the first patient last year, was designed primarily to assess safety. And so far, the procedure appears to be safe. By the end of the year, the researchers said they expect to share the first data on whether the procedure restored any vision for the patients.

golden rice opposition

• those opposed say the project is deeply flawed. • they point out that there are multiple recourses for malnutrition planned and currently in place, that are cheaper, do not require GMOs and that make golden rice unnecessary. • for example, UNICEF employs a vitamin A supplementation program that improves a child's survival rate by 12-24% at a cost of only a few cents per dose • friends of the earth (a non-governmental environmental organization ) stated that golden rice produces too little betacarotene to eradicate VAD, at 1.6 micrograms per gram of rice and 10% of the daily requirement of vitamin A. the amount of golden rice needed for sufficient vitamin A intake would be too great in comparison to the rice available in developing countries. • another point of opposition to the project stems from questions regarding the motives of the Golden Rice Project and its ties to several large biotech industries. • is it a ploy to enhance public support for GMOs, which could take funding away from cheaper, more realistic solutions? or are they out to make a profit? (those involved with the Golden Rice Project vehemently reject that their ties to biotech companies undermines their integrity.) • then there are concerns about the employment of the Golden Rice Project such as the cost to set up, technology transfer, the accessibility of the project, the sustainability and credibility of the rice, and stable support from governments.

gene modification of embryos (germline)

• when gene editing is used in embryos, or in gametes (sperm or eggs), it is called germline modification. • also known as "inheritable genetic modification" or "gene editing for reproduction," these alterations would affect every cell of the person who developed from that gamete or embryo and would be inherited by all future descendants. • assuming there is widespread adoption of the technology, it is possible that the genetic makeup of entire generations could permanently be altered. • there is broad agreement among many scientists, ethicists, policymakers and the public that while germline editing has enormous promise, its use should be restricted to research until the safety of the technology has improved and the ethical issues have been addressed. • scientists have concerns about the possibility of permanent harm to genetically modified individuals and their descendants as well as concerns about exacerbating social inequality, and conflict. • the clinical use of germline modification is prohibited in more than 40 countries and by an international treaty of the Council of Europe. despite this prohibition, in november 2018, a researcher shocked the world when he announced that he had created the world's first genetically modified people. he claimed that he utilized CRISPR technology to alter the CCR5 gene in a set of twins in an attempt to provide immunity to HIV. • this experiment has been widely condemned around the world • the Scientific Ethics Committees posted statements declaring their opposition to any clinical use of genome editing on human embryos, noting that "the theory is not reliable, the technology is deficient, the risks are uncontrollable, and ethics and regulations prohibit the action." • "opposed to any clinical operation of human embryo genome editing for reproductive purposes." • this development has resulted in enormous publicity regarding germline modification and has prompted a social debate about the use and governance of the technology.