Bio 3C Lab Quiz 3

what do restriction enzymes do

recognize and cut both strands of a specific sequence of DNA

what naturally makes restriction enzymes

Bacteria as an anti-viral response

what do we call viruses that infect bacteria

Bacteriophages

what is a recognized sequence is regards to restriction enzymes

a specific short DNA sequence that the enzyme can identify and bind to before cutting

recognized sequences are typically what?

palindromic/ an inverted repeat

EX: 5’C-C-G-C-G-G 3’

3’ G-G-C-G-C-C 5’

how can you tell if a cleavage product is sticky or blunt

sticky is if the cut is not the same place, blunt is if it is as the same place

in the photo top is blunt and bottom is sticky

DNA fragments generated by resection enzyme digest can be separated by what

gel electrophoresis in agarose or polyacrylamide gels

Recombinant DNA general steps

cleave plasmid vector with a restriction enzyme (commonly EcoRI), cleave the insert (DNA fragment) with the restriction enzyme, anneal and fragments and rejoin using the enzyme DNA ligase

how can eukaryotic genes be expressed in bacteria

via reverse transcriptase

what is cDNA

complementary DNA made from mRNA via reverse transcriptase

only thing that naturally makes reverse transcriptase

retroviruses

example use of reverse transcriptase

the creation of proinsulin cDNA from proinsulin mRNA, the cDNA is then transcribed and inserted into a recombinant plasmid and bacteria are transformed

when making cDNA why do we use mRNA instead of DNA

because bacteria lack the machinery to splice introns out of the primary transcript which it made out of RNA nucleotides

Steps of reverse transcriptase

an Oligo(T) primer (linked thymidine) binds to the mRNA at the 3’ end, then reverse transcriptase creates the cDNA following the mRNA template, once the cDNA is complete the mRNA is hydrolyzed, then terminal transferase adds nucleotides to the 3’ end of the single strand cDNA which allows for DNA polymerase to come in and create the secondary complementary strand leading to a double-stranded cDNA

— comes hand in hand with —-

DNA analysis, sequencing DNA molecules

how is DNA sequencing done

the Sanger Dideoxy Method which is the controlled termination of replication

Sanger Dideoxy Method described, not modern

four tubes each with the same template DNA to be sequenced, primer, DNA polymerase, and a different labeled ddNTP (dideoxy nucleoside triphosphate). DNA polymerase will do its thing but randomly it will add a ddNTP and the reaction will stop. Each tube will have a variety of fragment in it that can then be isolated and examined via gel electrophoresis. these fragment correlate to the position of the nucleotides in the complementary strand

what are ddNTP’s

dideoxy nucleoside triphosphate, they are called a 2,2’,3,3’-dideoxy analog. They do not have an “OH” at carbon 3 so DNA polymerase cannot add onto it and replication will not continue

how are ddNTP’s labeled

either radiolabled or fluorescently

how do you read a Sanger Sequencing gel?

Bottom to top, bottom is the 5’ end and top is the 3’ end of the complementary stand. The Template strand is then the paring of that strand

How is modern Sanger Sequencing different?

each ddNTP has its own colored fluorescent label to it so everything can be done it one tube and on the gel we can organize the sequence based on the fluorescent color

PCR stands for what

polymerase chain reaction

PCR components

pair of primers that hybridize with the flaking sequences of the target DNA sequence, all four dNTP’s, and a heat stable DNA polymerase (often Taq)

what is the PCR cycle

strand separation at 95°C, hybridization of primer around 54°C, DNA synthesis at 72°C, and this all repeated about 30-40 times

in PCR during hybridization of primers why do parent duplexes (original DNA strands coming back together) not form

because the primers are present in large excess

How can the quantity of individual mRNA transcripts be determined?

through quantitative PCR aka real-time PCR

qPCR stands for what

quantitative polymerase chain reaction

what does qPCR monitor

DNA amplification as the reaction progresses

in qPCR how is the reaction progression monitored

by an increase in fluorescent signal which is created by SYBR Green 1 dye which binds to the DNA. It fluorescences brighter in dsDNA than in ssDNA, so there is an increase in fluorescence as the PCR reaction continues and dsDNA is formed

During/after the qPCR cycle what is made

a qPCR curve

phases of a qPCR curve

lag, exponential, linear, and plateau

what can you find from a qPCR curve

the C_T which is the cycle threshold. This is the number of PCR cycles is takes for the fluorescence signals to cross a certain threshold and become detectable above background noise.

what does a low or high C_T mean

A lower C_T means there was more original template present so it reached the threshold faster, a higher C_T means there was less original template present so it reached the threshold slower

what are DNA microarrays used for

to investigate the pattern and level of expression of all genes in a particular cell or tissue (which is the transcriptome)

How is a DNA microarray set up

mRNA is isolated and then converted to cDNA in the presence of fluorescent dNTPs (different colors for different samples), then you separate the cDNA into single strands and add it into the wells of a microarray, each well in the microarray holds a 20nt DNA sequence called an oligonucleotide, the cDNA will bind to any complementary sequences and anything unbound will be washed away. The remaining bound parts will hybridize and be visible after a scan due to their fluorescence. The spots will glow green, red, yellow, or not at all.

in a DNA microarray after the wells are scanned what do the different fluorescent colors signify

Green: decreased expression of a particular gene

Red: increased expression of a particular gene

Yellow: high expression in both samples

None: no hybridization/binding

what does CRISPR stand for

Clustered Regularly Interspaced Short Palindromic Repeats

in CRISPR describe Cas9 and guide RNA

Cas9-guide RNA complex is what binds to the complementary sequence of the target gene, then Cas9 cuts both DNA strands

when Cas9 cuts the DNA what happens to the broken strands

they are repaired by the cell. When they are repaired scientists can disable the target gene to study its normal function. Or the target gene with a mutation can be repaired by providing a normal copy of the gene

what is sgRNA

single guide RNA, an engineered form of guide RNA that forms a complex with Cas9, it is about 100nt in length and has two regions

What are the two regions of sgRNA

Guiding region: part of CRISPR RNA (crRNA in nature) that is typically a 20nt region complementary to the target DNA sequence and it is customized for each target of interest

Scaffold region: tracrRNA in nature, forms a multi-hairpin loop structure (scaffold) that binds tightly to a crevice in the Cas9 protein, this region is typically the same for all sgRNA

In order for the Cas9 complex to cut DNA what must occur

It first has to bind to a common sequence in the genome called a PAM (protospacer adjacent motif), then the sgRNA unwinds a part of the helix and binds to its complement, then Cas9 can cut the DNA

How can Cas9 be altered in CRIPSR to achieve alternate functions

Cas9 can have one or both cutting domains deactivated and an enzyme can be fused onto it. The Cas9 complex than acts as a transport for that enzyme to a specific DNA sequence. An example of this is fusing deaminase onto it which mutates specific DNA bases such as converting Cytidine into Thymidine

Cas9 can also be fully deactivated and then altered to promote gene transcription. This is done by adding proteins that activate transcription. This can be done by directly adding the proteins, by first adding peptides to which these proteins are added, or by recruiting these proteins to the gRNA

Blotting techniques? Names, what they look for, how they probe?

SNOW DROP

Southern Blot- looks for DNA and probes with DNA

Northern Blot- looks for RNA and probes with DNA or RNA

Western Blot- looks for proteins which binds to antibodies

CRISPR stands for

Clustered Regularly Interspaced Short Palindromic Repeats

where was CRISPR found

in bacteria as a defense mechanism

what are the two steps in gene editing

cutting the double-stranded DNA at a desired location and directing DNA repair to produce a desired sequence change

what happens to a bacterial cell when chromosomal DNA is cut

the cell will die unless the cut is repaired

How can cells repair double stranded breaks in DNA

Non-homologous end joining (NHEJ): Specific proteins reconnect the ends of the double stranded break back together. This process can randomly insert or delete one or more based which can lead to mutations that disrupt gene function or expression.

Homology Directed Repair (HDR): Enzymes patch the break using donor template DNA. Researchers design the donor template DNA which can include a desired sequence added onto both ends called a homology arm. These homology arms match the sequence upstream and downstream of the cut. A complementary DNA strand in created during repair.

What did we personally use CRISPR-Cas9 to do in lab

cut bacterial chromosomal DNA at a specific location within the lacZ gene, then take advantage of the cell’s HDR to create a desired change in the lacZ gene. This change was due to the donor template DNA have a stop codon in it to disrupt gene function.

In the CRISPR lab what was the E. coli used for

The E. coli strain was HB101-pBRKan (often called pBRkan), this strain held the Cas9 enzyme and it carried a plasmid containing a kanamycin resistance gene, a naturally functional lacZ gene, and a gene for HDR that was controlled by an arabinose-inducible promoter (so only with arabinose present will bacterial takes be able to take up donor template DNA to preform HDR and fix the double strand breaks)

What is Cas9

a protein nuclease that uses an RNA guide to cut a specific sequence of DNA

what was our Cas9 in the CRISPR lab engineered to cut

only at the lacZ gene

what is the lacZ gene

gene that codes for the enzyme β-galactosidase (β-gal) which is part of the lac operon. It allows for E. coli to break down lactose, specifically it can also hydrolyze a sugar known as X-gal which when hydrolyzed leads to a blue pigment. Through this we can use blue-screening to look for blue colonies and determine is the lacZ gene is functional

what does Cas9 need in order to cut

a guide RNA that will bring it to the specific target DNA sequence

in the CRISPR lab how did we transform the E. coli

We had two different plasmids with different contents but both had a spectinomycin (SPT) resistance gene

pLZ Donor (pD): has the donor template DNA that can be used by HDR which has a stop codon to stop lacZ’s function

pLZ Donor Guide (pDG): has the donor template DNA and a sequence that codes for the sgRNA, once the bacteria has this plasmid it can transcribe the sgRNA gene then the sgRNA can guide Cas9 to cut at the lacZ gene

in the CRISPR lab what two plasmids are present and what is in each

HB101-pBRKan plasmid with kanamycin resistance, lacZ gene, and Cas9 gene.

pLZDonor or pLZ Donor Guide with spectinomycin resistance, donor template DNA and/or not sgRNA gene)

In the CRISPR lab when picking out colonies from the starter plates, what type of starter plates did we have and what was in them

Starter Plate IX: which has IPTG (for the I) to allow expression of the lac operon (lacZ transcription), and X-gal (for the X) which is the chromogenic substrate of β-galactosidase and results in blue colonies. No arabinose so no HDR

Starter Plate IX/ARA: which has IPTG (for the I) to allow expression of the lac operon (lacZ transcription), and X-gal (for the X) which is the chromogenic substrate of β-galactosidase and results in mostly blue colonies. Arabinose allows for activation of HDR

what is IPTG

Isopropyl β-D-thiogalactopyranoside, which is an analog of allolactose and used to induce expression of the lac operon by binding to the repressor protein so that DNA Polymerase can bind to the promoter and the lacZ gene is able to be transcribed

In the CRISPR lab what do these plates under these conditions look like?

• E. coli + pLZDonor (pD) on IPTG/X-gal Starter Plate (IX)

• E. coli + pLZDonorGuide (pDG) on IPTG/X-gal Starter Plate (IX)

• E. coli + pLZDonor on IPTG/X-gal/Arabinose Starter Plate (IX/ARA)

• E. coli + pLZDonorGuide on IPTG/X-gal/Arabinose Starter Plate (IX/ARA)

• Blue colonies, no cutting of the lacZ gene

• Cutting of the lacZ gene, but no arabinose so no repair, so no colonies (chromosomal DNA when cut and not repaired dies)

• Blue colonies, no cutting of the lacZ gene

• White colonies, lacZ gene is cut but donor template DNA is inserted and results in the stopping of the lacZ gene so no β-galactosidase can hydrolyze X-gal and make blue pigment

CRIPSR. Once we have the transformed bacteria and want to grow them what do we plate them on and why?

On plates with spectinomycin so that only bacteria that as taken in the pD or pDG plasmid will survive and grow

Why did the starter plates have kanamycin on them?

to ensure that only E. coli of the HB101-pBRKan strain with the pBRKan plasmid would grow

if you grew HB101-pBRKan on a plate with arabinose and then moved them what would happen

if grown on arabinose they will keep the enzymes needed for HDR even if moved, but their daughter cells will not produce those HDR enzymes unless exposed to arabinose

We can use CRISPR for two diseases, what and how?

Sickle-cell anemia and transfusion dependent beta-thalassemia which both use Exa-cel. Exa-cel stands for exagamglogene autotemcel. This cuts out the BCL11A gene, which prevents the production of fetal hemoglobin, and it edits the enhancer region. Once the BCL11A edited gene is put back in it undergoes less transcription so the transcription of fetal hemoglobin can occur. As a result there is more fetal hemoglobin with two alpha and two gamma subunits instead of two alpha and two beta subunits as in adult hemoglobin.

what does ELISA stand for

Enzyme Linked Immunosorbent Assay

what is immunology

the study of the immune system

what are the two main branches of the immune system

Innate Defenses: Nonspecific and have a variety of factors such as normal microbiota, mucous membranes, phagocytic cells, fever, and antimicrobial substances

Adaptive Defenses: Specific to each pathogen and involve the production of antibodies. Antibodies location and attache themselves to their target and facilitate the destruction and removal of pathogens, they are highly specific to their targets/antigens

How are animals involved with antibodies

Animals can be injected with a specific antigen and their body will make antibodies against that specific injected antigen which is located in their serum. We can harvest and isolate that antibody.

What are primary antibodies

Antibodies that recognize and bind to antigens

What type[s] of antibodies does ELISA use

primary and secondary antibodies

How can we produce secondary antibodies

Same idea as primary. We inject animals with the human primary antibody, they will make antibodies against the primary antibody which we can then harvest and isolate

What are secondary antibodies

antibodies that bind to the primary antibody which is already attached to the antigen. They are often enzyme-linked

what is another term for an enzyme linked secondary antibody

a labeled secondary antibody

In our ELISA lab was the enzyme that the secondary antibody was linked to and its substate

the enzyme is HRP, horseradish peroxidase. And the substate is TMB, 3,3’,5',5’-tetramethylbenzidine

What is HRP and what is its substrate/ does to it

horseradish peroxidase, the enzyme linked to the secondary enzyme in the ELISA lab. its chromogenic substrate is TMB: 3,3’,5,5’-tetrametylbenzidine, which gets oxidized and forms a blue product

what does TMB stand for/do

3,3’,5’,5’-tetramethylbenzidine, it is the substrate for the enzyme linked to the secondary antibody, it is light sensitive

what type of ELISA did we use and what does that mean

we used an indirect ELISA. This means we start with antigen bound to the wells, then the primary antibody is added and binds to the antigen, then the secondary enzyme-linked antibody is added which binds constant chain of the already bound primary antibody, substrate is added and then colored product is formed

What is direct ELISA

sandwich ELISA. When primary antibody is first bound to the well then antigen is added which binds to the primary antibody, then the enzyme-linked antibody specific for the antigen is added and binds, substrate is added and colored product is formed

What is the point of a blocking buffer in ELISA

used to stop any non-specific binding by using an irrelevant protein or by using a non-ionic detergent

in our ELISA lab what blocking buffer did we use, where do we find this?

a non-ionic detergent called Tween-20, found in our wash buffer

in our ELISA lab what kid did we use to test for what

we used a kit from Bio-Rad to determine if mock parents have HIV viral proteins in their blood

what do actual HIV texts look for

anti-HIV antibodies since virion numbers will be low in the serum but antibody levels will be high

in our ELISA lab what was in the wash buffer and what did it do

Has 10% Tween-20 which is an non-ionic surfactant that removes anything unbound while also binding to the plastic (polystyrene) wells and saturating biding sites as to avoid nonspecific binding

in our ELISA labs what are the wells made out of and why

Polystyrene which binds to proteins by hydrophobic interactions

in ELISAs why is it important to not have non-specific binding

if antibody is bound non-specifically to the well the that means it will be present even if antigen isn’t present which can lead to a false positive result

in ELISAs why do we wash between each step

to remove anything that is unbound and prevent previous components from reacting in later steps and possibly causing a false positive

What happens in an ELISA when you add a primary antibody if the antigen is and isn’t present

if the antigen is present and you add primary antibody, the antibody will bind specifically to the antigen creating an antigen-antibody complex that will not wash off

if the antigen is not present and you add primary antibody there will be no binding and no creation of an antigen-antibody complex so the primary antibody will be washed away

What are three other immunoassays other than ELISA

radioimmunoassays, western blot, lateral flow assays

In order for an ELISA to determine if there is a positive result what do we need to add

a chromogenic substrate in the end to produce a colored positive result

in the ELISA lab what antigen, primary antibody, and secondary antibody did we use exactly

Antigen: chicken gamma-globulin (purified egg yolks)

Primary antibody: polyclonal rabbit antibody raised against chicken gamma-globulin

Secondary antibody made from goats immunized with rabbit IgG

In ELISA what can lead to false positives or negatives

False positive can result from recent vaccinations. False negatives can result from recent immunosuppression, or administering the test too soon after infection with HIV

Antibody structure?

Heavy inside chain and light outside chain, variable (top four section) region and constant (bottom eight section) region, F_C and F_ab region. The part of the antibody that binds to and recognizes the antibody is called the epitope.

Antigen defintion

a substance that causes the body to produce specific antibodies or sensitized T cells

what are immunoglobulins

globular proteins, specialized proteins called plasma cells in the immune system

what is valence in terms of antibodies

the total number of antigen binding sites on the antibody

How are B cells activated

Antigen binds to receptor, B cells take in the antigen, B cells then present pieces of the antigen on the surface via MCH Class II molecules, Helper T cells then binds to the MCH Class II molecules, Helper T cells release cytokines, those cytokines then fully activate the B cells which then differentiate into plasma or memory cells

what are the classes of immunoglobulins, what determines that class

IgG, IgM, IgA, IgD,IgE. This is determined by the amino acid sequence of the constant region of the heavy chains

what are the protective mechanisms of binding antibodies to antigens

Agglutination: Antibodies bind to multiple antigens at once and clump pathogens together to make them easier for immune cells to detect and remove since they are grouped and not spaced out

Opsonization: Antibodies coat a pathogen and act like a tag that allows for immune cells (like macrophages) to recognize the antibody and phagocytose the pathogen more easily

Neutralization: Antibodies bind to viruses or toxins and block them from entering or damaging cells. They inactive the threat without destroying it.

Antibody-dependent cell-mediated cytotoxicity: Antibodies attach to infected or abnormal cells, then immune cells (like natural killer cells) recognize those antibodies and kill the targeted cell

Activation of Complement: Antibodies trigger the complement system which is a group of proteins in the blood that leads to cell lysis, increased inflammation, and increased opsonization

understand the idea of this graph

the first exposure takes longer to produce antibodies and makes less of them. the second exposure creates antibodies way faster and produces way more of them.

what are common blocking agents besides the Tween-20 used in our ELISA

non-fat dry milk, gelatin, and bovine serum albumin