FIU Biotech Summer Exam 2 - Dr Sharp

What is a Plasmid

- Circular piece of DNA found in bacteria

- Extrachromosomal DNA (1 to 4kb size)

- Can replicate independently

- Used as a vector

Calcium Chloride Transformation

- Process of inserting Foreign DNA into Bacteria

- Very Inefficient

Electroporation

- Brief pulse of high voltage, creates tiny holes in bacteria that allow DNA to enter

- Rapid, require fewer cells, more efficient

Selection

- Facilitates identification of recombinant bacteria from non-recombinant bacteria

-Prevents growth of non transformed bacteria

Antibiotic selection

Does not select for plasmid containing foreign DNA vs recirculized with no DNA

X-Gal Blue-white selection

- When LacZ gene is interrupted, not beta-galactosidase (success)

- White = clones with recombinant plasmid

- Blue = clones with no Recombinant DNA (recircularized)

Multiple Cloning site (MCS)

Recognition site for restriction enzymes

What is a DNA Library

Collection of cloned DNA contained in plasmids

Types of DNA Library

- Genomic

- Complementary (cDNA)

What type of DNA do genomic libraries use

- Chromosomal DNA from tissue

Disadvantages of genomic libraries

- Introns are cloned (Majority of genomic DNA is introns)

- Difficult to search, time-consuming

- no information on gene expression

What type of DNA do cDNA libraries use

- mRNA from tissue

- Makes DNA from RNA

Advantage of cDNA libraries

- Introns not cloned

- information on gene expression

Disadvantages of cDNA libraries

- hard to make if source is not available

What is used for library screening to identify a gene of interest

- Colony hybridization

- Bacteria with recombinant DNA grown on plate

What type of filter is used for colony hybridization

Nylon or nitrocellulose

Downstream processing that relies on ability of most proteins to bind specifically and reversible to ligands

Affinity chromatography

What is most likely to occur during upstream processing of proteins using biotechnology

Expression of the protein in the cell

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

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

Reverse transcription PCR

What laboratory technique utilizes a probe with a 5' end reporter and 3' end quencher to study gene expression

Real time PCR

Why are proteins so fragile

Due to weak hydrogen bonds

Assume the human genome project was not completed but you wanted to clone growth hormone from humans, what sequence would you use to design PCR primers

Use mouse or rat sequence because genes of these species are similar to humans

Why might normal chemotherapy not be as effective as glycoprotein therapy

Chemotherapy does not exclusively target cancer cells. It would target other cells which could potentially compromise the immune system

What additional enzymes studied in CH3 would need to be mass produced for recombinant DNA technology

Restriction enzymes

How do you lose cells and denature their DNA in colony hybridization

- Alkaline solution

- Bake filter/expose it to UV

- single stranded DNA binds to filter as a result

What do you incubate the filter with

-A probe tagged with radioactive nucleotide / fluorescent dye

- probe depends on what is known about gene of interest

What does the probe bind to

-complementary sequence on filter via hydrogen bonding

- definition of hybridization

How do you see the results from colony hybridization

- Expose to X-ray film / digital camera

- detects fluorescent probe

- compare to original

If gene sequence has NOT been cloned in another species but something is known about the protein, what can be done

- Work backwards

- Design probe based on DNA of the protein

Who developed PCR

Kary Mullins in 1983

What does PCR do

Amplifies sequence of DNA in a short period of time

Ingredients of PCR

-Target DNA

-dNTP's

- MgCl2

- Buffer

- Taq polymerase

- F/R primers

Where is PCR done?

thermocycler

What is denaturation?

- unfolding of proteins

- 94 - 96C

What is annealing (hybridization) in PCR?

Primers bond, 52 - 58C

Extension (PCR)

-heating (70 - 75 degree C) to increase the rate of replication by DNA polymerase

-DNA polymerase replicates each strand to produce more DNA

- cycles repeated 20 - 30 times

Why cant you use DNA polymerase from bacteria that lives optimally at 37C

It would denature at higher temperatures

Advantage of cloning PCR products

Faster and more effective than DNA libraries

What species is Taq polymerase derived from

Thermus aquaticus that thrives in hot springs

Applications of a gene of interest

- Making dna probes

- STudying gene expression

- detection of viral and bacterial infections

- diagnosis of genetic conditions

- detection of trace amounts of DNA tissue found at crime scene

- detection of DNA from fossilized tissue

Disadvantage of cloning PCR products

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

Great trick

Taq polymerase puts a single adenine nucleotide on the 3' end of all PCR products

How many primers for DNA sequencing

1

What is the chain termination sequence known as

Sanger Method

Why is DNA sequencing important?

to determine the sequence of nucleotides of the cloned gene

WHat did original Sanger method have

- 4 seperate reaction tubes

- Radioactively labeled nucleotides

- 200- 400 nucleotides per reaction

What does new Sanger method use

Capillary electrophoresis

-1 reaction tube

- fluorescent dye nucleotide

- produces electropherogems

- produces greater than 600 nucleotides

- 900 bp sequence

2nd GS with pyrosequencing

- ROche 454 commercial system

- utilizes pyrophosphate in its biochemistry

- chemiluminescent (light releasing) RXN's

uses ATP sulferase

NGS (2nd GS) Post Light Sequencing

-Ion Torrent PGM

-utilizes release of H+ on semiconductor chip

3rd GS with single molecule reads

-oxford nanopore technologies

-minlon reads single molecules

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

-sensor detects changes in ionic current at nanopore

What causes overlapping peaks in electropherogram

Contamination

What does DNA polymerase release after integrating a nucleotide

Hydrogen (H+) and pyrophosphate

What is the name of the pyrosequencing machine

Roche 454

What does ATP sulferylase do

Converts PPi to ATP in presence of 5 phoshosulfate

What is the substrate for conversion of Luciferin to oxyluciferin

ATP (generates light)

WHat is the name of post light sequencing machine

Ion torrent PGM

What does MinIon do

Reads single molecules

Who developed southern blotting

Ed Southern in 1975

What is southern blotting?

Used to determine gene copy number

- gene mapping

- gene mutation detection

- PCR product confirmation

Southern blotting

Digests chromosomal DNA into small fragments with restriction enzymes

What is Northern blotting used for

- Studying gene expression

- analyzing mRNA produced by a tissue

- is similar to southern blotting

Where will sperm be

Epididymis segment A

House keeping gene

a gene that is expressed at similar levels in all cell types providing an essential function such as Actin

What is Reverse Transcription PCR used for

used to study mRNA levels when level of detection is below that of Northern (gene expression)

- uses PCR to amplify cDNA from specific gene if interest

What is real time or quantitative PCR (qPCR) used for

Quantify amplification rxns as they occur in real time

- needs special thermal cyclers that use a laser

-each tube contains a Fluorescent dye containing probe with quencher or DNA binding dye

WHat are the two approaches used for qPCR

- Both start with mRNA -> cDNA

1. Taqman probes

2. SYBR green

TaqMan probes in qPCR

- complimentary to specific regions of Target cDNA between forward and revers primers for PCR

- contain reporter 5' end of probe and can emit fluorescence light when excited by the laser

SYBR green

binds double stranded DNA and as

more double stranded DNA is copied with each round of qPCR there are more DNA copies to bind SYBR Green which increases amount of fluorescent light emitted

What is fluorescence in situ hybridization (FISH)

- CHromosome location of gene and copy number

- identify which chromosome contains a gene of interest

- creates a karyotype

What does fluorescence on more than one chromosome indicate

Gene is found more than one chromosome (Gene duplication

What is (FISH) used to analyze

- Genetic disorders

- determine which cells in a particular organ or tissue are expressing the particular mRNA

What are proteins made of

Chains of amino acids

- have specific molecular weights

- have electrical charges that cause them to interact with other molecules

WHat does structure an function of a protein depend on

Protein folding

What happens if a protein is folded incorrectly

Can lead to disease, protein function is lost

Who described Alpha helices and beta sheets in 1951

Pauling and Corey

What is primary protein structure

Sequence of linked amino acid

What is the secondary structure of a protein?

Folded chains of amino acids (beta and alpha helix sheets)

What is the tertiary structure of a protein?

3 dimensional, secondary structures cross linked

- determines protein function

What is the quaternary protein structure?

Unique, 3 dimensional, build of several polypeptide

What is glycosilation

Post translational modification, carbs added to protein

How can post translational modification and glycosilation affect protein activity

- increase solubility

- protein orientation

- extended life

What can glycoproteins be used for

- to destroy lymphoma cancer cells

- combined with nano particle loaded with chemotherapy drug

What is a bioreactor?

cell system that produce biological molecules

Biotech production of target proteins

• Proteins produced via microbial or mammalian cell culture

• Complicated and time-consuming process

• Large batches of the desired protein produced in bioreactors

What are some applications of proteins in healthcare

- Treatment of damaged corneas

- screening molecules associated with disease

Protein application in industry

- food processing

- textiles

- detergents

- bioremediation

What does directed molecular evolution do

- mimics the process of natural selection to evolve proteins

- induces mutations randomly (mutagenic PCR)

Difference between molecular evolution and site directed mutagenesis

- directed molecular evolution -> random mutations

- site directed mutagenesis - > predefined alterations

What is upstream processing

Includes actual processing of protein in a cell

what is down stream processing

Involves purification and verification

What is first of upstream processing

Selecting for protein source (Bacteria, fungi, plants, mammals, insects)

advantages of bacteria in upstream processing

- genetics well understood

- unlimited quantities

- fermentation tech well understood

Disadvantages of bacteria in upstream processing

- Foreign protein must be refolded

- proteins cannot be folded in ways needed for humans

- some proteins are inactive in humans

- foreign proteins form insoluble clumps-inclusion bodies

What are fungi used for in upstream processing

- source of wide range of proteins (used in animal feed and beer )

- used as hosts for engineered proteins

WHat are advantages of fungi in upstream processing

- capable of post translational modifications

- allow proper folding of proteins

What are plants used in upstream processing

To produce specific proteins that do not occur naturally

WHat is a great choice for biotech production proteins

Tobacco

Disadvantage of plants in upstream processsing

Not all proteins are expressing in plants

- have cell walls

- glycosilation different from animal cells

What are animal bioreactors used for

- Use for monoclonal antibody production

- Mice are injected with an antigen and mouse

secretes the desired antibody and the antibody is

then purified