Plasmids & DNA Cloning

What are the three types of cloning?

I) Organismal

II) Theraputic

III) Molecular (or gene) cloning

Organismal cloning

is the process of creating a genetically identical copy of an entire organism, often through techniques such as somatic cell nuclear transfer. E.g., Dolly the sheep

Theraputic cloning

involves creating embryonic stem cells for use in medical treatments and research, allowing for the development of tissues or organs that are genetically matched to the patient.

Molecular/gene cloning

Isolation of a defined piece of DNA (usually containing a gene) and producing many copies of that sequence before putting it back in bacteria for further application

What makes cloning genes possible?

I) Ability to ‘cut and paste’ DNA (restriction enzymes)

II) Ability to isolate & amplify cut-and-pasted DNA

Restriction enzymes

Part of restriction/modification system of prokaryotes

Modification portion of restriction enzymes

chemical modification of bacterial DNA by methylation

Restriction portion of restriction enzymes

ezymatic cutting of unmodified DNA rendering it non-functional; restriction endonucleases

T/F: MEthylation of DNA affects base pairing and protein interaction

False; only effects protein interaction

Three classes of restriction endonucleases

Type I, Type II, and Type III; types I & III have both modification and restriction functions (not useful) → type II have separate restriction and modification enzymes (very useful)

Who first discovered restriction enzymes (endonucleases)?

Smith & Wilcox (1970)

Type II restriction endonucleases

• sequence specific recognition site

• that cleave DNA at specific sites, leaving blunt or sticky ends. They are widely used in molecular biology for cloning and DNA manipulation.

• recognition sites = palindromic

5’ overhang

A type of DNA end formed after cleavage by restriction enzymes, characterized by one strand extending further than the other, which facilitates ligation with complementary overhangs.

3’ overhang

A type of DNA end formed after cleavage by restriction enzymes, characterized by one strand being shorter than the other, allowing for ligation with complementary sequences.

Blunt ends

A type of DNA end created by cleavage that results in both strands of DNA being of equal length, preventing the formation of sticky overhangs.

Restriction mapping

A technique used to determine the locations of restriction enzyme cut sites within a DNA molecule by analyzing the sizes of the resulting fragments.

Lambda phage chromosome

The genome of the lambda bacteriophage, a virus that infects bacteria, consisting of linear double-stranded DNA that can integrate into the host genome.

Gel electrophoresis

A laboratory method used to separate DNA, RNA, or proteins based on their size and charge by applying an electric field to a gel matrix.

Recombinant DNA

A form of DNA that is created by combining DNA from different organisms, resulting in a sequence that contains genes from two or more sources. (insert & plasmid)

Plasmid

A small, circular piece of DNA that replicates independently of chromosomal DNA in bacteria and can carry genes for traits such as antibiotic resistance. Have been modified to be used as cloning vectors.

Cloning vector

A DNA molecule used to carry foreign genetic material into a host cell for replication and expression.

Bacterial transformation

The process by which bacteria take up foreign DNA from their environment, allowing for genetic alteration and experimentation.

What is the purpose of a cloning vector?

The purpose of a cloning vector is to introduce foreign genetic material into a host cell, facilitating its replication and expression in order to produce proteins or study gene function. However, does not produce proteins; done by expression vector.

Components of cloning vectors

• Restriction enzymes recognition sequences

• Bacterial origin of replication

• Selectable marker

Restriction enzyme recognition sequences

are specific nucleotide sequences that restriction enzymes recognize and cleave, allowing for the insertion of foreign DNA into cloning vectors.

Bacterial origin of replication

is a specific DNA sequence that allows for the initiation of DNA replication within a bacterial cell, ensuring that the cloning vector can replicate independently of the host's chromosomal DNA.

Selectable marker

is a gene that confers a trait onto a cell, such as antibiotic resistance, allowing for the identification and selection of cells that have taken up the cloning vector.

Cohesive compatible ends

are short, single-stranded overhangs created by certain restriction enzymes that facilitate the specific ligation of DNA fragments during cloning.

Complementary sticky ends

are single-stranded DNA overhangs generated during restriction digestion that can anneal to each other, enhancing the efficiency of DNA fragment ligation in cloning.

Directional cloning

is a cloning strategy that uses two different restriction enzymes to create compatible ends, allowing DNA fragments to be inserted into a vector in a defined orientation.

Three desirable features of directional cloning

• only insert-DNA fragments possessing two different compatible ends will be effectively inserted into vector

• This selective mechanism ensures that the fragments are oriented correctly, promoting consistent expression and functionality in the resulting recombinant DNA.

• Vector cannot relegate itself

What two methods can transform a restriction enzyme site into blunt ends?

DNA polymerase (5’ overhang) & exonucleases (3’ overhang)

Amplification

is the process of increasing the number of copies of a specific DNA sequence, typically using techniques such as polymerase chain reaction (PCR) or other cloning methods.

Transformation

is the introduction of foreign DNA into a host cell, enabling the uptake and expression of that genetic material, often used in genetic engineering to create recombinant organisms.

How can you tell if cloning/transformation has been successful?

• Antibiotic selection

Antibiotic selection

is a method used to identify successfully transformed cells by applying antibiotics, which kill cells that have not taken up the plasmid containing an antibiotic resistance gene.

Blue-white screening

is a technique used in cloning to identify recombinant bacteria by their color change; typically, colonies with the inserted gene will appear a different color compared to non-recombinant colonies. (No insert = blue, insert = white

Component cells

are bacterial cells that have been prepared to take up plasmid DNA during the transformation process.

What are the two steps to make cells ‘competent’ to take up DNA?

I) Dilute salt solution on ice (solidifies membrane changes)

II) Heat shock (causes fluid influx causing uptake of DNA)

Genomic Library

is a collection of cloned DNA fragments that represent the entire genome of an organism. It allows researchers to study specific genes and sequences within the genome.

CDNA

complementary DNA synthesized from an mRNA template. It is used in various applications such as gene cloning and expression analysis.

Expression vectors

vectors that have been furnished with sequences capable of directing efficient transcription & translation of trans genes

Trans genes

genes that have been introduced into an organism's genome through genetic engineering. They can be used for various purposes including research, agriculture, and medicine.

Shine-Dalgarno sequence

A ribosomal binding site in bacterial mRNA that facilitates the initiation of translation by aligning the mRNA with the ribosome.