Biotech Exam 2

What does PCR stand for?

Polymerse Chain Reaction

Who developed PCR, and what year?

Kary Mullis in 1983.

What is the definition of PCR?

Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time.

All of the following is the process for what?

- Target DNA to be amplified is added to a tube, mixed with nucleotides (dATP, dCTP, dGTP, dTTP), buffer with MgCl2, and DNA polymerase.

-Paired set of forward and reverse primers are added -short single stranded DNA oligonucleotides (18-22 nucleotides long).

-Reaction tube is placed in an instrument called a thermocycler.

Process of PCR.

What is the cofactor of PCR and what is the purpose of a cofactor?

Mg. The purpose of the cofactor is to speed up a reaction. DNA polymerase without Mg won't do its job.

Heat to 94-96 degrees C.

Denaturation.

(hybridization) in which primers H bond with complementary bases at the end opposite ends of target sequence at 52-58 degrees C.

Annealing.

(elongation) DNA pol copies target DNA at 70-75 degrees C.

Extension.

What are the three stages that occur within a thermocycler?

1. Denaturation

2. Annealing.

3. Extension

At which step of the thermocycler do you want the primers to anneal?

Annealing.

Thermocycler will take DNA through a series of reactions called a?

PCR cycles.

How many times are PCR cycles repeated, and are they doubled?

20-30 times. And yes they double each time.

True or False: You want a primer-dimer or hairpin loop?

False you do not want a primer-dimer or hairpin loop.

Advantage of PCR:

Amplify millions of copies of target DNA from small amount of starting material in short period of time.

Isolated from a species known as Thermus aquaticus the thrive in hot springs.

Taq DNA polymerase.

What are the applications of DNA?

-Making DNA probes

-Studying gene expression

-Detection of viral and bacterial infections

-Diagnosis of genetic conditions

-Detection of trace amounts of DNA from tissue found at crime scene

-Detection of DNA from fossilized tissue.

What is a disadvantage of Cloning PCR products?

Need to know something about the DNA sequence that flanks the gene of interest to design primers.

Is rapid and effective compared to using DNA libraries?

Cloning PCR products.

Great trick:

Taq polymerase puts a single adenine nucleotide on the 3' end of all PCR products. This knowledge is used to the researchers advantage: T vector that has single stranded thymine on each end so can complementary base pair with the adenine in the PCR products.

Chain termination sequencing is

Sanger method

Sanger method was developed by? And is important to determine?

Frederick Sanger and colleagues in 1977. The sequence of nucleotides of a cloned gene.

Reaction tubes for Sanger method require?

-Single primer annealing to denatured DNA template.

-All 4 dNTPs.

-DNA polymerase and buffer solution.

-Dideoxynucleotide (ddNTP) which has a 3' H instead of 3' OH and deoxyribose so it cannot form a phosphodiester bond with the incoming nucleotide and so gets terminated.

What is the percent for a terminator?

There is a 1% chance.

Had four seperate reaction tubes and each contained the same DNA, radioactively labeled single primer, all 4 dNTPs, and a small amount of one ddNTP per tube, DNA pol.

Original Sanger method.

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.

Original Sanger method.

-Fragments are separated on polyacramide gel.

-Read the gel from bottom to top as individual nucleotides.

Original Sanger method.

-The sequence generated from the reaction is complimentary to the sequence on the template strand in the vector.

-Sequencing 200-400 nucleotides per reaction.

Original Sanger method.

What are some key difference between PCR/Sequencing?

-Only one primer instead of two.

-Besides having normal nucleotides you are going to have terminator.

-The ratio of these terminators is about 1%.

-For PCR you need one double stranded DNA, but for sequencing you already have to have hundreds of millions of copies. In order to figure out sequencing you already have had to have done PCR. When you do DNA sequence you are not trying to make more copies.

Is a gel that does a really good job at resolving fragments that are super similar in size. An agarose gel does not achieve the same results. It is toxic however until it solidifies.

Polyacryamide.

Sanger method using _____________ enables greater than 600 nucleotides to be sequenced per reaction.

Capillary electrophoresis.

-Was very helpful for completing human genome project.

-Procedure: use only 1 reaction tube instead of 4.

Sanger method using Capillary electrophoresis.

True or False: Capillary electrophoresis allows multiple runs as long as you flush polymers through the capillaries?

True.

True or False: By having the terminators in the same tube you can get away with using 4 tubes.

True.

All of the following occur under?

-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.

Sanger method using Capillary electrophoresis.

1st generation sequencing is?

Capillary electrophoresis.

All of the following pertains to?

-Roche 454 commercial system.

-Utilizes pyrophosphate in its biochemistry.

-Chemiluminescent (light releasing) RXNs.

NGS (2nd GS) with Pyrosequencing.

True or False: Pyrosequencing works off of Pyrophosphate?

True.

Steps to Pyrosequencing:

Do DNA extraction then go and fragment it with restriction enzymes. In this type of sequence you can only sequence fragments that are 200-400 nucleotides. Hydrosequencing is about 200 with ion-torment its about 400 these reactions are occurring in microcells. You might have millions of wells b/c you have a bunch of nucleotides.

All of the following pertains to?

-Ion torrent PGM.

-Utilizes release of H+ on semiconductor chip.

-DNA-Ions-Sequence.

-Nucleotides flow sequentially over Ion semiconductor chip.

-One sensor per well per sequencing reaction.

-Direct detection of natural DNA extension.

-Millions of sequencing reactions per chip.

NGS (2ndGS) Post light sequencing.

All of the following pertains to?

-Oxford Nanopore technologies.

-Minlon reads single molecules.

-10+kb reads, error rate approx. 5%.

-Sensor detects changes in ionic current at nano pore.

3rd GS with single molecule reads.

When DNA goes from gel to Nitrocellular paper.

Blotting.

Used to determine gene copy number, gene mapping, gene mutation detection, PCR product confirmation and was developed by?

Southern Blotting. Ed Southern.

All of the following pertain to?

-Digest chromosomal DNA into small fragments with restriction enzymes.

-Fragments are seperated by agarose gel electrophoresis.

-Gel is treated with alkaline solution to denature the DNA.

-Fragment are transferred onto a nylon or nitrocellulose filter (called blotting).

Filter (blot) is incubated with a labeled probe and exposed to film by autoradiography or digital camera.

-Number of bands on film represents gene copy number.

Southern Blotting.

What are the tools in our tool box?

-Restriction enzymes.

-Six-base pair cutters.

-Gel-electrophoresis.

-Plasmid vectors, different type of plasmids.

-Transform

-PCR

-DNA sequencing.

What is one downfall of Souther blotting?

It is very time consuming.

What are some of the steps to Southern blotting?

We take DNA and digest it with RE, then we do Gel electrophoresis. We get a smearof different sized fragments that are distinct, then we treat the gel with an alkaline solution bc it will denature the DNA from double stranded to single stranded, then we blot.

After blotting you expose the DNA to UV light and what that does is make it permanent. Then you get the probe to bind to our DNA of interest.

What is a probe?

It is a small stretch of single stranded DNA that has complimentary sequence. The number of bands represents the number of copies.

What is the order of the materials from bottom to top used in Southern blotting?

Transfer buffer, platform, Saran Wrap, whatman paper, gel, membrane, Whatman paper stack, paper towel stack, weight.

What is the purpose of Southern Blot? And what carries the DNA during blotting?

To locate our gene of interest. The buffer.

Technique involves analyzing mRNA produced by a tissue.

Northern blotting.

All of the following pertain to

-RNA is isolated from a tissue of interest.

-RNA is seperated by gel electrophoresis and blotted into a nylon membrane, and hybridized to a labeled DNA probe.

-Exposed bands on autoradiograph show presence of mRNA for gene of interest as well as size of mRNA.

-Can compare and quantify amounts of mRNA present in different tissues.

-Basic method is similar to Southern blotting.

Northern blot.

Western blot is for _______, and Eastern blot _________

Proteins, does not exist.

What is being expressed at that moment in time. Then run it out on the gel.

mRNA extraction.

Used to study gene expression. Technique involve analyzing mRNA produced by a tissue.

Northern blot analysis.

Accessory organs to the males reproductive tract.

Seminal vesicles.

Outside of the testes, short term storage for spam, vas deference is for long term storage.

Epididymus.

Expressed evenly in all tissues.

Housekeeping gene.

Nitrocellulose filter paper.

Foil.

Gives you more information bc it looks at levels of a particular gene.

Northern blot.

You are just trying to locate a particular gene of interest.

Southern blot.

Used to study mRNA levels when levels of detection is below that of Northern blot.

Reverse transcription PCR.

The following procedure refers to?

-Isolate mRNA and use __________ to make double stranded cDNA.

-Use PCR to amplify region of cDNA with set or primer specific for gene of interest.

-Run agarose gel to separate amplified cDNA fragments.

-Determine expression patterns in the tissues your comparing.

-Amount of cDNA produced in _______ reflects amount of mRNA and level of gene expression.

Reverse transcription PCR.

RTPCR stands for?

Reverse transcription PCR.

If there is not enough mRNA for us to be able to see with our eyes we use what?

RTPCR.

An antimicrobiano protein.

Defensin.

Can quantify amplification rxns as they occur in real time.

Real time or quantitative (qPCR).

Need special thermal cycles that use a laser to scan a beam of light through the top or bottom of each PCR rxn. 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.

Real time quantitative (qPCR).

Light emitted via fluorescence correlates with amount of PCR product amplified. Light is captured 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.

Real time or quantitative (qPCR).

A dye that binds to double stranded DNA.

Cybergreen.

A single stranded DNA that on one end has a reporter that when you hit it with light it will fluores is called?

Taqman probe.

True or False, when the reporter and quencher are on the DNA strand you don't get a light.

True. Light appears when they are not present.

Two approaches, both start with RNA to cDNA.

Real time or quantitative (qPCR).

Studying gene expression.

Why would you prefer taqman probe over SYBR green?

Bc SYBR green does not care where it binds to.

Are complimentary to specific regions of target cDNA between forward and reverse primers for PCR.

Taqman probes.

Containreporter located at 5 prime end of probe and can emit fluorescent light when exited by the laser without the quencher which is attached to the 3 prime end.

Taqman probe.

Binds to double stranded DNA, and as more double stranded DNA is copied with each round of qPCR there are more DNA copies to bind _______ which increases the amount of fluorescente light emitted.

SYBR green.

Forward and reverse PCR primers bind to denatured target DNA. Taqman probe with reporter and quencher dye binds to target DNA between the primers When the probe is intact, emission by the reporter dye is quenched.

Hybridization.

As DNA polymerase extends the forward primer, it reaches the Taqman probe and cleaves the reporter dye from the probe. Released from the quencher the reporter can now emit light when excited by a laser.

Extension.

Is when the instrument and software package is analyzing what is happening in every single tube, anything below the instruments and software wont care too much about.

Threshold cycle.

High expression and low expression have to do with what?

Have to do with when you cross the threshold cycle mark, mRNA to cDNA has to do a lot with the threshold. It's not about how tall the line is, but at what point was the threshold line passed.

True or False, Taqman probe binds to any gene?

False, Taqman probe only binds to our gene of interest.

Chromosome location of gene and copy number. Identify which chromosome contains a gene of interest.

Fluorescence in situ hybridization (FISH).

The following procedure pertains to?

-Chromosomes are isolated from cells and spread out on glass microscope slide.

-DNA or RNA probe for gene of interest is labeled with fluorescent dye and incubated with slides.

-Probe will hybridize with complementary sequences on chromosomes on slide.

-Slide is washed and then exposed to fluorescent light.

-Wherever probe ha bound to the chromosome, it is illuminated to indicate the presence of the probe binding.

-Perform Karyotype to determine which chromosome shows fluorescence.

Fluorescence in situ hybridization (FISH).

Is associated with fancy Karyotypes. Basically you get a chromosome that is stuck in metaphase lyse the cells, spread out the chromosomes, use single stranded DNA and RNA probe that has a dye attached to it. You can determine which cell is expressing a particular gene you are interested in.

Fluorescence in situ hybridization (FISH).

To determine which chromosome are homologous they are aligned according to their length and fluorescent patters of their chromatids and used to create a karyotype.

-Is used to analyze genetic disorders. Used to determine which cells in a particular organ or tissue are expressing the particular mRNA.

Fluorescence in situ hybridization.

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. Fluorescent in situ hybridization

D. Reverse transcription PCR

E. None of the above.

D. Reverse transcription PCR.

What laboratory technique is used to identify which chromosome contains a gene of interest when generating a karyotype?

A. Fluorescence in situ hybridization

B. Southern blot

C. Western blot

D. Reverse transcription PCR

E. None of the above.

A. Fluorescence in situ hybridization.

What is a challenge of biotechnology?

Understanding and controlling the protein folding in the manufacturing process.

Complex molecules built of chains of amino acids. Have specific molecular weights. Have electrical charge that causes them to interact with other molecules.

Some are hydrophilic - water loving

Some are hydrophobic - water fearing.

Proteins.

The structure and function of a protein depends on

Protein folding.

What are some of the dangers of a mis folded protein?

If a protein if folded incorrectly, the desired function of a protein is lost and a mis folded protein can be detrimental. FE, Alzheimer's, cystic fibrosis, mad cow, forms of cancer and even some heart attacks.

Described two regular 2nd structures. Alpha helices and Beta sheets. Structures are fragile, hydrogen bonds are easily broken.

Pauling and Corey 1951.

What are the four protein folding structure levels?

Primary, secondary, tertiary, quarternary.

Is the sequence in which amino acids are linked together.

Primary structure.

Occurs when chains of amino acids fold or twist at specific points forming new shapes due to the formation of hydrogen bonds between hydrophobic amino acids.

Secondary structure.

Most common shapes of a protein are?

Alpha helix, Beta sheets.

Amino acids form right handed spiral as hydrogen bonds stabilize the structure linking an amino acids nitrogen atom to oxygen atom of another amino acid.

Alpha helix.

Hydrogen bonds link nitrogen and oxygen atoms forming sheets that are parallel (chains run in same direction or anti-parallel (chains alternate in direction).

Beta sheets.

3 dimensional polypeptides are formed when secondary structures are cross linked. Determines the function of the protein.

Tertiary structure.

Are unique, globular, three-dimensional complexes built of several polypeptides.

Quarternary structures.