Biotech Exam 2 BSC4422 FIU

CHAPTER 3: Identify & Clone Gene of Interest

DNA Libraries

Define DNA Libraries

Collection of cloned DNA fragments,

from a particular organism,

contained in plasmid vectors, within a host bacteria.

Screen the library to?

Pick out different genes of interest.

2 Types of DNA Libraries:

Genomic DNA Libraries

&

Complementary DNA Libraries (cDNA Libraries)

Genomic DNA Libraries (3)

1. Chromosomal DNA,

from tissue of interest,

is -isolated- & -digested-,

with a restriction enzyme which produces many fragments [that include the entire genome]

2. Plasmid Vectors digested with same enzyme^

3. Ligase used to ligate chromosomal DNA fragments within plasmid vector

Recombinant Plasmid Vectors used to?

Transform Bacteria

(so each bacteria will contain a single recombinant plasmid)

Disadvantages of Genomic Libraries (3)?

1. Clones introns!!

In addition to exons, introns cloned!

Majority of genomic DNA in eukaryotes is Introns (NON-CODING PIECES of DNA)

2. Hard & Time-consuming

Many organisms have large genomes, so seeking gene of interest is difficult

3. Not provide information on all levels of gene expression

Description of cDNA Libraries?

mRNA from tissues of interest is Extracted ~~~>>

cDNA Libraries

How to make double stranded DNA from mRNA? (3)

1. Short Linker double stranded DNA sequences, which contain restriction enzymes recognition sites ADDED to the ENDS of the cDNA.

2. Cut with restriction enzymes, cut vector, & ligate fragments to create:

3. Recombinant vectors

Disadvantage of cDNA libraries (1)

Can be difficult to make IF a source tissue (with an abundant amount of mRNA for the gene) is not available.

Advantage of cDNA libraries (3)

1. Does not clone introns!

2. Create & Screen isolated genes (that are only expressed under certain conditions in a tissue)

3. Collection of actively expressed genes from isolated mRNA.

Library Screening:

COLONY HYBRIDIZATION (9)

1. Bacteria with recombinant DNA grown on agar plate

2. Nylon or Nitrocellulose filter placed over plate

3. Treat filter with alkaline solution

-DOES 2 THINGS:

Lyses Cells & Denatures DNA

4. Bake filter or UV exposure

5. Incubate filter with a PROBE that is tagged with either:

-Radioactive nucleotide or Fluorescent dye

6. HYBRIDIZATION: Probe binds by H bonding to complementary sequence on the filter

7. Filter washed (to remove excess unbound probe)

8. Filter exposed to either:

X-ray film or digital camera

to detect fluorescent probe

9. Use film or picture to compare to the OG agar plate to identify which colonies contained the recombinant plasmid with gene of interest.

Colony Hybridization:

Type of probe used depends on?

What is already known about the gene of interest

Library screening rarely results in cloning of full-length gene.

What do scientists look for? (3)

1. Get small pieces of the gene

2. Scientist look for overlapping sequences

3. They look for START (AUG) and STOP codons to know when full-length of gene is obtained

Polymerase Chain Reaction (PCR)

Developed in 1983 by Katy Mullis (1944-2019).

Technique:

Making copies or amplifying a specific sequence of DNA in a short period of time.

Process of PCR (4)

1. Target DNA amplified is added to a tube

2. Mixed with nucleotides (dATP, dCTP, dGTP, dTTP), buffer with MgCl2, and DNA polymerase

3. Add Paired set of Forward and Reverse Primers (short single stranded DNA oligonucleotides (18-22 nucleotides long)).

4. Reaction tube placed in THERMOCYCLER

PCR Cycle

Thermocycler takes DNA through series of reactions

3 Stages:

1. Denaturation- 94-96 C

2. Annealing- (Hybridization) 52-58 C; primers H bond with complementary bases at the opposite ends of target sequence

3. Extension- (Elongation) 70-75 C; DNA polymerase copies target DNA

End of 1 Cycle, DNA is?

Doubled

Cycles are repeated?

20-30 times

Advantages of PCR (2)

1. Amplify millions of copies from small starting material in short time

2. Calculate # of copies of target DNA starting with 1 molecule using Equation: 2^N

where N is number of cycles

How many copies of DNA will 22 cycles generate?

2^22 = 4, 194, 304

Applications of PCR: (6)

1. Making DNA probes

2. Studying gene expression

3. Detection of -viral & bacteria- infections

4. Diagnosis of genetic conditions

5. Detection of trace amounts of DNA from tissues at CRIME SCENE

6. Detection of trace amounts of DNA from fossilized tissue

Advantage: Cloning PCR Products is _______ & _______ compared to DNA libraries:

Rapid and Effective

Disadvantage of Cloning PCR Products (1)

Need prior knowledge about the DNA sequence that flanks the gene of interests to DESIGN primers

Taq DNA polymerase

1. Isolated from species Thermus aquaticus that thrive in hot springs

2. Puts a single adenine nucleotide (A) on the 3' end of ALL PCR products

(T vector has single stranded Thymine (T) on each end so can complementary base pair with the A).

DNA Sequencing: Sanger (2)

1. Chain termination sequencing

2. Developed by Frederick Sanger 1977

Sanger Sequencing is Important to?

determine the sequence of nucleotides OF CLONED GENES

Sanger Sequencing Method (4)

1. Reaction tube containing:

2. Single primer annealing to denatured DNA template

3. All 4 dNTPS (dATP, dCTP...)

4. Dideoxynucleotide (ddNTP)

Sanger: Dideoxynucleotide has?

3' H instead of 3'OH on the deoxyribose

so it cannot form a phosphodiester bond with incoming nucleotides and so gets terminated

Sanger Sequencing: OG method: 4 Separate Reaction tubes containing: (5)

1. same DNA

2. radioactively labeled single primer

3. all 4 dNTPS

4. small amount of ddNTP (per tube)

5. DNA polymerase

Sanger Sequencing: OG method (5)

1. 4 separate reaction tubes

2. Over time, ddNTP will be incorporated into all positions of newly synthesized strands creating fragments of varying lengths that are terminated at the ddNTP

3. Fragments separated on polyacryamide gel

4. Autoradiography used to identify radioactive fragments

5. Read from BOTTOM to TOP as individual nucleotides

Sanger Sequencing: OG method: Sequence generated from reaction is complementary to?

sequence on template strand in vector

Sanger Sequencing: OG method: Sequencing how many nucleotides per reaction?

200-400

Read sequence from _____ to _____ fragments

smallest to largest

High Throughput Computer Automated Sequencing: Sanger

using capillary electrophoresis,

enables greater>600 nucleotides per reaction

*very helpful for HUMAN GENOME PROJECT

High Throughput Computer Automated Sequencing: Sanger Procedure (

1. Uses 1 reaction tube instead of 4

2. ddNTPs labeled with different fluorescent dye

3. Laser stimulated dye which emits different wavelengths of light

4. emitted light is collected by detector (feeds to computer)

5. Electropherogram: computer CONVERTS light patterns to reveal nucleotide sequence

Next-Generation Sequencing (1st)

Using capillary electrophoresis

Next-Generation Sequencing (2nd NGS & Pyrosequencing): (2)

1. Roche 454 commercial system

2. Utilizes PYROphosphate &

chemiluminescent (light releasing RXN)

Next-Generation Sequencing (2nd NGS & Post Light Sequencing): (2)

1. Ion Torrent PGM

2. Utilizes release of H+ on semiconductor chip

Next-Generation Sequencing (2nd NGS & Post Light Sequencing):

DNA->...

DNA--> ION --> 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

Next-Generation Sequencing (3rd NGS with Single Molecule Reads): (4)

1. Oxford Nanopore Technology

2. Minlon reads SINGLE MOLECULES

3. 10+kb reads, Error Rate: 5%

4. Sensor detects changes in ionic current at nanopore

Southern Blotting: By? When?

Ed Southern 1975

Southern Blotting used to? (4)

Determine:

1. Gene Copy #

2. Gene Mapping

3. Gene Mutation

4. PCR Product Confirmation

Southern Blotting Procedure:

1. Digest chromosomal DNA into small fragments with restriction enzymes

2. Fragments separated by agarose gel electrophoresis

3. Gel is treated with alkaline solution

DOES 2 THINGS:

-lyses cells and denatures DNA

4. BLOTTING: Fragments transferred to nylon OR nitrocellulose filter

5. Filter (Blot) baked or exposed to UV light [to permanently attach DNA]

6. Filter (Blot) incubated with probe

7. Exposed to film OR autoradiography OR digital camera

8. Number of bands on film = gene copy #

Southern Blotting: Number of Bands on film =

Gene Copy #

Studying Gene Expression: Northern Blotting

Technique involves analyzing mRNA produced by a tissue

Northern Blotting

Basic method similar to Southern Blotting

-RNA is isolated form tissue of interest

-RNA is separated by gel electrophoresis

-Blotted on nylon membrane

-Hybridized to labeled DNA probe

-Exposed bands on autoradiography show presence of mRNA for gene of interest & SIZE of mRNA

-compare and quantify amounts of mRNA present in diff tissues

Northern Blotting: Exposed bands on autoradiography show:

Presence of mRNA (of gene of Interest)

AND

Size of mRNA

Northern Blotting Analysis

1. Seminal vesicles

2. Kidney

3. Epididymis Segment A

4. Epididymis Segment B

Reverse Transcription PCR (RT-PCR)

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

Reverse Transcription PCR (RT-PCR) Procedure (4)

1. Isolate mRNA and use Reverse transcriptase to make double stranded cDNA

2. Use PCR to amplify region of cDNA with set of specific primers

3. Run agarose gel to separate

4. determine expression patterns in tissues you're comparing

Reverse Transcription PCR (RT-PCR): Amount of cDNA produced for gene of interest reflects?

amount of mRNA and level of gene expression

Real Time or Quantitative PCR (qPCR)

Can quantify amplification rxns as they occur in real time

Real Time or Quantitative PCR (qPCR) needs:

special thermal cyclers that use lasers to scan a beam of light through the top OR bottom of each PCR rxn

Real Time or Quantitative PCR (qPCR): Reaction tube:

contains EITHER

-fluorescent dye containing probe with quencher

-DNA binding dye that emits fluorescent light when illuminated by laser

Real Time or Quantitative PCR (qPCR): Light emitted via fluoresc dye correlates with?

amount of PCR product amplified

Real Time or Quantitative PCR (qPCR) Procedure

1. Light is captured by the detector which feeds computer to provide readout on amount of fluoresc

2. Readout is then plotted and analyzed to quantitate the number of PCR products produced after each cycle

Studying Gene Expression: 2 Approaches?

1. Taqman

2. SYBR-green

Studying Gene Expression: 2 Approaches; Both start with: (2)

1. RNA ---> cDNA

2. qPCR

Studying Gene Expression: Taqman (2):

1. Taqman probes are complimentary to specific regions of target cDNA between forward and reverse primers for PCR

2. Taqman probes contain reporter located at 5' end of probe and can emit fluoresc light when excited by the laser without the quencher which is attached to 3' end of probe

Studying Gene Expression: SYBER-green (1):

SYBER-green binds double stranded DNA and as more double stranded DNA is copied with each round of of qPCR there are more DNA copies to bind SYBER-green which increases the amount of fluoresc light emitted

Fluorescence in Situ Hybridization (FISH)

Chromosome location of gene and copy number

Which chromosome contains gene of interest

FISH Procedure: (6)

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

2. DNA and RNA probe for gene of interest is labeled with fluoresc dye and incubated with slides

3. probe will hybridize

4. slide is washed and exposed to fluoresc light

5. wherever probe has bound to the chromosome, it is illuminated to indicate the presence of the probe binding

6. perform karyotype to determine which chromosome shows fluoresc

FISH Procedure Determination

To determine which chromosome is homologues, they are aligned according to length and fluoresc pattern of their chromatid and used to create a karyotype

FISH is used to (2):

1. analyze genetic disorders

2. determ which cells in the particular organ or tissue are expressing particular mRNA

CHAPTER 4: Protein in Biotechnology Products

Understand and control protein folding in the manufacturing process

Define PROTEINS

complex molecules built of chains of amino acids

PROTEINS have? (2):

1. Specific molecule weights

2. Electrical charge that causes them to interact with other molecules:

-hydroPHILIC [water loving]

-hydroPHOBIC [water hating]

Protein Folding

Structure and function depends on protein folding

If Protein is Folded Incorrectly?

the desire function is lost and can be detrimental

Incorrect folding leads to? (5)

Diseases:

-Alzheimer's

- cystic fibrosis

- mad cow

-forms of cancer

-heart attacks

Most common shapes: Alpha helices & Beta sheets

Pauling and Corey 1951

Two regular 2nd structures

Structures are fragile, H bonds easily break

Protein Structure (4 Levels)

1. Primary: sequence in which amino acids are linked together

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

3. Tertiary: 3 dimensional polypeptides are formed when secondary structures are cross linked

(DETERMINES FUNCTION)

4. Quaternary: unique, globular, 3D complexes built of several polypeptides.

Most Common Protein Shapes (2):

1. Alpha Helix:

am ac form right handed spiral as H bonds stabilize the structure linking an amino acids nitrogen atom to oxygen atom to form another am ac

2. Beta Sheet:

H bonds link nitrogen and oxygen atoms forming sheets that are parallel or antiparallel

Protein Structure: Glycosylation

Post-translational modifications wherein carbohydrate units are added to specific locations on proteins

Eukaryotic Cells have more than?

100 post-translational modifications occuring

Protein Structure: Glycosylation: Change can affect proteins activity by: (3)

1. increasing solubility

2. orient proteins into the membrane

3. extend the active life of a molecule in an organism

Example of Glycoproteins used as treatment of disease and how?:

1. Used as a NEW way to target and destroy B lymphoma cancer cells

*How?

Glycopro is combined with nanoparticle loaded with chemotherapy drug

targeting cancer cells with receptors

glycopro attached nanoparticles loaded with chemotherapy agent, the effective dose of the drug is increased while protecting normal tissues

Use of Protein in Manufacturing: (3)

Time-tested technology

1. Beer-brewing

2. Wine-making

3. Cheese-making

Recombinant DNA technology made it possible to produce specific proteins on demand: (3)

1. Enzymes

2. Hormones

3. Antibodies

Biotech Production of Target Proteins (3):

Proteins produced via microbial or mammalian cell culture

Complicated and time-consuming

Large batches of the desired proteins produced in bioreactors

Bioreactors?

Cell system that produce biological molecules

Biotech Drugs and other medical applications: Within Bioreactors: (3)

1. cells stimulated to produce proteins thru precise culture conditions that include balance of temp, oxy, acidity

2. At times, the proteins are isolated from cultures, tested at every step of purification and formulated into pharmaceutically active products

3. must comply with FDA regulations at all stages

Applications of Protein in Healthcare (2)

1. Treatment of damaged corneas

-help regenerate and repair damaged eye tissues and improve vision

2. Screening molecules associated with disease

-identify early predictor biomarker proteins

EX: PSA prostrate specific antigen test

Applications of Protein in Industry (4)

1. Food processing

2. Textiles and leather goods

3. Detergents

4. Bioremediation

Protein Engineering **

design and build new proteins in lab

-invent proteins that are tailored to specific applications

Protein: Directed Molecular Evolution **

method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids toward user-defined goal

** Protein Engineering by Directed Molecular Evolution

can induce mutations randomly into genes (mutagenic PCR)

then select bacterium with protein product enzyme that has HIGHEST activity

Protein Engineering by Site Directed Mutagenesis

introducing specific PREDEFINED alterations in the am ac sequence through a process called SITE DIR MUtag...

Producing Proteins (2 major)

long, painstaking process

2 major phases:

1. UPSTREAM Processing

includes the actual expression of the protein in the cell

2. DOWNSTREAM Processing

involves purification of the protein and verification of the function; a stable mean of preserving the protein required

*Choices made during upstream processing can simplify downstream processing

Protein Expression: Upstream Processing

selecting cell to be used as protein source

Protein Expression: Upstream Processing: Bacteria (5)

Fermentation process are well understood

cultured in large quantities in short period of time

relatively easy to alter genetically

increase level of production of bacterial protein by introducing additional copies of relevant gene to the host cell via recombin DNA

most common bacterial species used: E. coli

Protein Expression: Upstream (2)

1. Bacteria - majority of proteins synthesized naturally by E. coli are intracellular [in the cell]

2. E coli can be genetically engineered to produce the desired protein in the form of a fusion protein

Protein Expression: Upstream: Bacteria grown:

Genetically engineered bacteria can be grown in large scale fermenters (anaerobic)

or

bioreactors (aerobic)

Protein Expression: Upstream FUNGI:

USED in products like

-animal feed

-beer

-hosts for engineered proteins

*fungi are eukaryotic and capable of post-translational modifications to allow for proper folding of proteins

Protein Expression: Upstream Plants

used for protein expression

-plants can be genetically engineered to produce specific proteins that do NOT occur naturally

Protein Expression: Upstream Plants Process:

encourages rapid growth and reproductive rates in plants

-tobacco plant great for protein production

-when gen material integrated, a million new plant protein factories can fill fields

-disadvantage: not all proteins can be expressed in plants; have cell walls; process of glycosylation is slightly different from animal cells

Protein Expression: Upstream Mammalian Cell culture systems= challenging

-Nutritional requirements are complex

-mammalian cells grow slowly

-easy to contaminate

*best choice for proteins destined to be used in humans

Protein expression: animal bioreactors production systems

Use for monoclonal antibody production

-mice are injected with antigen , then secretes desired antibody, then antibody is purified

What is an Antibody?

Proteins produced in reaction to antigens of invading viruses, bacteria, etc.

Antibodies combine with & neutralize antigen, protecting organism.

-helps living things against diseases