Genetic Engineering and Biotech

Biotechnology

Use of living organisms to create products useful to humans (e.g. vitamins, antibiotics, foods, enzymes)

Genetic engineering

Deliberate modification of an organism’s genome sequence

Recombinant DNA technology

Procedures used to combine genetic material from multiple sources and create DNA sequences that are otherwise not found in biological organisms

Recombinant DNA

DNA from two or more sources (may be members of same domain or different domains)

5 steps of the basic mechanism of gene cloning

(1) isolate plasmid DNA from bacterial cell (2) isolate DNA of interest (3) insert the gene of interest into the plasmid DNA, forming recombinant DNA (4) insert the recombinant DNA into a bacterium, such as E. coli (5) culture the bacteria, growing many identical copies

Restriction enzymes

Type of endonuclease that is an important tool that allows scientists to cut up DNA at specific sites

Why are restriction enzymes naturally produced by bacteria?

To defend against viral infection

Most restriction enzymes recognize…

Restriction sites (i.e. recognition sequences)

Restriction sites

Short nucleotide sequences, between 4 and 8 nucleotides long

Restriction sites are usually…

Palindromes

Palindromes

Symmetrical nucleotide sequences running in opposite directions on two strands

Restriction enzymes cut DNA in such a way that they leave…

Sticky ends

DNA ligase

Seals recombinant strands together by catalyzing phosphodiester bonds between the single-strand breaks

How is a recombinant plasmid transferred to a host?

By treating them with calcium chloride and then applying a heat shock, making the cells competent; electroporation may also be used

What is protoplast fusion (removing the cell walls of bacteria, which allows them to fuse) and direct microinjection with a “gene gun” used for?

To introduce foreign DNA into a host cell

… are the most common prokaryotic hosts, and … are the most common eukaryotic hosts

E. coli; S. cerevisiae (a type of yeast)

4 steps for constructing dsDNA from ssRNA using RT

(1) beginning with a ssRNA molecule, apply a short poly-T primer (2) add RT and the four nucleotides (A, C, G, T) (3) add an enzyme called RNaseH to cut up the RNA and regenerate the RNA primers (4) add DNA polymerase and DNA ligase to synthesize a new strand

RT can only add nucleotides to an existing…

3’ hydroxyl group

Cloning vector

Provides a means for transferring a gene of interest to a host organism during the cloning process

3 characteristics of a “good” cloning vector

An origin of replication, a selectable marker, and unique restriction sites

Why is it important that cloning vectors have an origin of replication?

DNA polymerase begins DNA replication at the origin

Why is it important that cloning vectors have a selectable marker?

To allow scientists to easily identify and isolate the bacteria with the plasmid that contains the gene of interest; can be an antibiotic resistance gene or a color marker

Why is it important that cloning vectors have unique restriction sites?

The vector must allow the insertion of the gene of interest via RT; polylinkers or multicloning sites (MCSs)

What is the most important vector used in cloning techniques?

Plasmids (e.g. pBR322, pUC19)

Why are plasmids popular?

They can be easily transferred from one organism to another via conjugation or transformation

Plasmids are…

Autonomous/self-replicating

Bacteriophage

Virus that infects bacteria

Cosmid

Hybrid between a plasmid and a phage; carries larger DNA fragments

Artificial chromosomes

Synthetic chromosomes that contain fragments of DNA integrated into a host chromosome; carries larger DNA fragments

What vectors were used in the Human Genome Project?

Artificial chromosomes

DNA analysis

Used to determine the location of genes, the differences in genes, and where and when genes are expressed

Polymerase chain reaction (PCR)

Technique to quickly and easily make many copies (amplification) of even a very small amount of DNA from any source; faster than standard cloning techniques

PCR involves incubating the target DNA with…

Primers, thermostable DNA polymerase, and deoxyribonucleotide triphosphates

What DNA polymerase is typically used in PCR?

Taq polymerase

Thermocycler

Used in PCR; an instrument that cyclically increases and decreases the temperature of the solution

Each cycle of PCR… the DNA content

Doubles

End-point PCR

Used for diagnostic processes; involves making a large number of copies of a DNA segment

Real-time PCR (qPCR)

Allows researchers to monitor the amount of DNA in a sample over time; allows scientists to track how much DNA is in a sample over the course of a reaction

Gel electrophoresis

Separates nucleic acids and proteins on the basis of their size and electrical charge, using a polymer gel

Common gels for gel electrophoresis

Agarose or polyacrylamide

Nucleic acids have a… charge due to their…

Negative charge; phosphate backbones

In gel electrophoresis, nucleic acids migrate through the electric field towards the…

Positive elctrode

The… the DNA, the… the distance towards the positive electrode the DNA will migrate on the gel

Longer; shorter

In gel electrophoresis, DNA sequences are separated by…

Size

In gel electrophoresis, the migration rate is…

Inversely proportional to the log of its molecular weight

In gel electrophoresis, what dye is used to stain the DNA?

Ethidium bromide

Restriction fragment length polymorphism (RFLP)

Used for genetic fingerprinting, genome mapping, localization of genes, paternity testing, and microbial classification

5 steps of RFLP

(1) isolate DNA of interest (2) amplify the DNA using PCR techniques (3) cut the DNA up with restriction enzymes, producing restriction fragments (4) use gel electrophoresis to separate the restriction fragments according to their lengths on agarose (5) use computers to analyze results

RFLP relies on the premise that…

Every organism’s restriction sites will be located at slightly different locations on the genome, so the specific pattern of restriction fragments on gel will differ from person to person (DNA fingerprint)

Blotting

Combines electrophoresis and hybridization to detect specific DNA fragments

Southern blotting

Uses radioactive DNA probes and autoradiography to identify the presence of a specific gene

Autoradiography

Method for detecting radioactively labeled molecules

5 steps of southern blotting

(1) cut the DNA with restriction enzymes (2) separate the DNA fragments by size on an agarose gel (3) place a sheet of nitrocellulose paper on top of the gel and apply pressure from the top to transfer DNA from gel to paper (4) place paper in heat-sealed bag with radioactive probe (5) wash the filter and place it on X-ray film to produce an image of DNA bands

In an autoradiograph, if the DNA contained the gene of interest, … will be visible

DNA bands

Western blotting involves… and Northern blotting involves…

Proteins; RNA

Genomic library

Collection of an organism’s entire genomic DNA, which is stored in a population of identical vectors, each of which has a fragment of DNA

4 steps of creating a genomic library

(1) extract DNA from cell of interest (2) use restriction enzymes to cut DNA into fragments of a specific size (3) insert the fragments into a vector (4) insert the vector into a host organism

When is a genomic library used?

When the gene of interest resides on a chromosome that has not been sequenced

Site-directed mutagenesis

Technique used to introduce mutations into the genome and reverse genetics

Reverse genetics

Molecular genetic technique in which scientists analyze the phenotypic effects of altering a DNA sequence in order to understand the gene’s functions

Protoplast fusion

Most widely seen in fungi; cell walls of multiple cells removed, electric shock applied, and fusion occurs; allows scientists to combine the most desirable characteristics of different species

Medical applications of genetic engineering

Production of desirable proteins, gene therapy, vaccines

Scientists have engineered bacteria to produce massive amounts of…

Insulin

a-, b-, and y interferons

The body’s “natural antiviral molecules;” used to treat viral infections

Gene silencing

Use of siRNAs, which bind to mRNA and then destroy it by recruiting a RISC

Gene editing

Through CRISPR; a pre-designed “guide RNA” sequence binds to DNA portion that is to be edited, then guides Cas9 to to the site to cut the dsDNA

Subunit vaccines

Contain antigenic components of a pathogen, previously grown in GM yeast

DNA vaccines

Have the genetic sequences for the pathogen’s antigens

Agricultural applications of genetic engineering

Production of genetically modified plants, production of biopesticides

Tumor-inducing (Ti) plasmid

Bacterial plasmid with Ti genes removed, used to insert genes into plant genomes

Biopesticides

Naturally produced agents that kill pests

Bacillus thuringiensis (Bt)

Gram-positive bacterium that produces pesticide toxin; used by Monsanto

Structural genomics

Study of the physical nature of genomes

Functional genomics

Study of the function of genomes

Comparative genomics

Study of the differences among the genomes of different organisms

Bioinformatics

Study of the genome using computers

Annotation

Used to determine the location of genes on a genome

Annotation searches for…

Open reading frames (ORFs)

Open reading frames (ORFs)

Relatively long sequences of codons that are not interrupted by a stop codon (100+ codons in prokaryotes)

In annotation, computers can look for… at the 5’ end and… at the 3’ end

Ribosomal binding sites; characteristic terminator sequences

Coding sequences

ORFs presumed to encode proteins

Paralogs

Two genes that consist of similar sequences and are found in the same genome

Paralogs usually arise due to…

Gene duplication events

Orthologs

Two genes that consist of similar sequences and are found in different organisms

Motifs

Short patterns of amino acids known to have a specific function

Examples of DNA binding motifs

Helix-turn-helix, zinc finger, leucine zipper

In silico analysis

Analysis carried out by a computer that allows researchers to further examine bioinformatics

DNA microarray analysis

Allows scientists to observe the pattern of DNA expression for thousands of genes at a time

5 steps of DNA microarray analysis

(1) researcher collects mRNA molecules present in a cell (2) RT used to convert mRNA into cDNA labeled with fluorescent dye (3) cDNA placed onto microarray chip (4) cDNAs bind to complementary DNA on the chip (5) intensity of fluorescence measured

In DNA microarray analysis, the… areas represent the genes with the most… genes in the cell

Brightest; “active”

Functional proteomics

Study of protein function that determines what actually occurs in the cell

Structural proteomics

Study of 3D structures of proteins

2D gel electrophoresis can separate proteins based on… and…

Charge; mass

In 2D gel electrophoresis, how are proteins separated by charge?

Isoelectric focusing; proteins move in the gel until they reach the point where their net charge is 0

In 2D gel electrophoresis, how are proteins separated by molecular mass?

Gravity; smaller proteins will migrate farther down into the gel

SDS polyamide

Used to simply separate proteins based on their molecular weight

Metabolomics

Study of the metabolome (set of small-molecule metabolites present within a cell at a given time) to allow researchers to assess the physiological status of a cell at any given point in time

Lipidomics

Study of the lipidome (entire lipid profile of the cell) to allow researchers to assess the impact of the environment on a cell’s membrane

Comparative genomics has led to the creation of…

New therapy targets and vaccines

Comparative genomics has assisted in developing a therapy for…

Leprosy; by comparing the bacterial parasite that causes leprosy with that of TB