BCM6225 Cloning Pt. 2 & 3

Recombinant DNA technology
DNA cloning
Molecular cloning
Gene cloning

The transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid.

foreign host cell

The DNA of interest can then be propagated in a?

bacterial plasmids

Scientists studying a particular gene often use _______ to generate multiple copies of the same gene.

Plasmids

These are self-replicating extra-chromosomal circular DNA molecules, distinct from the normal bacterial genome.

True.

Plasmids and other types of cloning vectors were used by Human Genome Project researchers to copy genes and other pieces of chromosomes to generate enough identical material for further study.

T or F?

restriction enzymes

To clone a gene, a DNA fragment containing the gene of interest is isolated from chromosomal DNA using ____ and then united with a plasmid that has been cut with the same _____.

Recombinant DNA molecule

This is produced when the fragment of chromosomal DNA is joined with its cloning vector in the lab.

host cell DNA

Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the?

Dolly

This was the first mammal to be cloned from adult DNA.

Reproductive cloning

A technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal.

Somatic Cell Nuclear Transfer (SCNT)

This process allowed scientists to transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed.

chemicals or electric current

The reconstructed egg containing the DNA from a donor cell must be treated with ______ or _____ in order to stimulate cell division.

uterus

Once the cloned embryo reaches a suitable stage, it is transferred to the _____ of a female host where it continues to develop until birth.

True.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal.

T or F?

chromosomal or nuclear DNA

In the case of Dolly. only the clone's ______ or _________ DNA is the same as the donor.

mitochondria

Some of the clone's genetic materials come from the _______ in the cytoplasm of the enucleated egg.

aging process

Acquired mutations in the mitochondrial DNA are believed to play an important role in the?

Therapeutic cloning

This, also known as embryo cloning, is the production of human embryos for use in research.

Stem cells

The goal of therapeutic cloning is not to create cloned human beings, but rather to harvest _______ that can be used to study human development and to treat disease.

These cells are important because they can be used to generate virtually any type of specialized cell in the human body.

5 days

Stem cells are extracted from the egg after it has divided for how many days?

blastocyst

The egg at this stage of development is called a?

False. It destroys the embryo, so it raises a variety of ethical concerns.

The extraction process doesn't destroy the embryo, which doesn't raise a variety of ethical concerns.

T or F?

Gene cloning

This is the process of amplifying a specific piece of DNA via a bacterial cell.

Cloning vector

A replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell.

1. origin of replication
2. antibiotic resistance gene
3. cloning site

Cloning vectors have features that make it easier to insert DNA and select for presence of vector in cell namely (3):

origin of replication

In gene cloning, first, a cloning vector must contain an _________ recognized in the host cell so that it is replicated along with the DNA that it carries.

selectable markers

In gene cloning, second, it should carry _______ a.k.a traits that enable cells containing the vector to be selected or identified.

single cleavage site

In gene cloning, third, a cloning vector needs a single ________ for each of one or more restriction enzyme used.

Plasmid vectors

These are small DNA circles that are physically separated from chromosomal DNA and can replicate independently.

Linkers

These are synthetic DNA fragments containing restriction sites.

1. Isolation of DNA fragments (genes of interest) to be cloned

2. Insertion of isolated DNA into a suitable vector to form the recombinant DNA

3. Introduction of the recombinant DNA into a suitable host cell

4. Selection of the transformed host cells and identification of the clone containing the gene of interest

5. Multiplication/Expression of the Introduced gene in the host

6. Isolation of the multiplied gene copies/protein expressed by the introduced gene

7. Purification of the isolated gene copy/protein

7 major steps in cloning genes:

Plasmid

This cloning vector can clone DNA as large as 15kb through plasmid replication and introduced to bacteria through transformation.

Phage lambda

This cloning vector can clone DNA as large as 23kb through phage reproduction and introduced to bacteria through phage infection.

Cosmid

This cloning vector can clone DNA as large as 44 kb through plasmid reproduction and introduced to bacteria through phage infection.

Bacterial artificial chromosome

This cloning vector can clone DNA as large as 300 kb through plasmid reproduction and introduced to bacteria through electropulsation.

Electroporation/electropulsation

This process consists of electrical pulses that increase permeability of a membrane; creates microscopic pores in the plasma membrane called electropores.

1. can allow the easy entry of the recombinant DNA into the cell

2. should not destroy the recombinant DNA as a foreign DNA and degrade it

3. can stably maintain the recombinant DNA

4. the transformed host must not independently sustain outside the laboratory

5. should be easy to handle

6. should be available as a wide variety of genetically defined strains

7. should accept a range of vectors

7 characteristics of a good host cell for cloning:

deOR mutations

These mutations allow the easy entry of recombinant DNA across the host cell; it encodes for proteins or transporters that assist in the uptake of large DNA molecules.

Inclusion bodies

These are products of failed protein expression.

mutant hsdR

This is a member of the E. coli strain K family which recognizes a certain marker for the recombinant DNA (AACNNNNNNGTGC); an endonuclease.

active hsdM

This is a member of the E. coli strain K family which is a methylase that protects the recombinant DNA.

recA and recF

These are mutations that inactivate the recombination of gene of interests; both mutants.

Calcium chloride-mediated DNA transfer

This method of gene transfer involves transformation of prokaryotic host cells using calcium chloride.

Microinjection

This method of gene transfer involves the direct introduction of the recombinant DNA into the host cell.

Biolistic particle delivery system

This method of gene transfer involves coating the recombinant DNA with microscopic tungsten particles (microprojectiles), which are then accelerated on a macro-projectile by firing a gunpowder charge or by using compressed gas to drive the macro-projectile.

Sonoporation

This method of gene transfer involves the use of sound (typically ultrasonic frequencies) for the transfer of recombinant DNA into the target host cell.

Optical transfection

This method of gene transfer involves the host cell to be exposed to the highly focused laser beam for a small amount of time (tens of milliseconds to seconds), generating a transient pore on the membrane called photopore.

Impalefection

This method of gene delivery uses Nano materials, such as carbon Nano fibers, carbon nanotubes, nanowires, etc.

Protein expression

This refers to the mechanisms by which proteins are synthesized, modified, and regulated in living organisms; systems engineered to express multiple copies of a desired protein within a host cell.

transformation

Protein expression in bacteria wherein an expression vector is inserted into the host cell is called?

transfection

Protein expression in mammalian cells wherein an expression vector is inserted into the host cell is called?

Prokaryote

The ribosomes on this type of cell consists of the 30S and 50S subunit.

Eukaryote

The ribosomes on this type of cell consists of the 40Sand 60S subunit.

Prokaryote

The mRNA present in this type of cell is polycistronic mRNA.

Eukaryote

The mRNA present in this type of cell is monocistronic mRNA and is involved in post-transcriptional processing.

Prokaryote

This type of cell has the Shine-Dalgarno sequence on the mRNA transcript; first amino acid is formylated methionine.

Eukaryote

This type of cell observes cap-dependent and cap-independent translation.

Cap-dependent translation

This type of translation in eukaryotes involves a Cap structure and the Cap-binding proteins mediated translation.

Cap-independent translation

This type of translation in eukaryotes involves ribosomes binding to mRNA occurring through internal ribosome entry site (IRES) on mRNA.

Prokaryote

This type of cell has 3 initiation factors namely IF1, IF2, and IF3.

Eukaryote

This type of cell has more than 3 initiation factors.

Prokaryote

This type of cell has 3 elongation factors namely EF-Tu, EF-Ts, and EF-G.

Eukaryote

This type of cell has 2 elongation factors namely EF1 (alpha, beta, and gamma) and EF2.

Prokaryote

This type of cell has 2 termination factors namely RF-1 and RF-2.

Eukaryote

This type of cell has a termination factor called eRF-1.

1. Polypeptide folding into a globular protein mediated by chaperones.

2. Modifications of the amino acids present

3. Disulfide bridge formation or reduction

4. Protein modifications that facilitate binding functions:
Glycosylation
Prenylation of proteins
Acetylation of histones

5. Addition of functional groups that regulate protein activity: Phosphorylation, Nitrosylation, & GTP binding

5 post-translational modifications:

1. mammalian
2. insect
3. yeast
4. bacterial
5. algal
6. cell-free

6 types of expression vectors: