Plasmids and DNA Cloning

Organismal (or Reproductive) Cloning

producing a second, genetically identical copy of an organism (e.g. Dolly)

Therapeutic (or Embryo) Cloning

production of human embryos for research purposes only

Molecular (or Gene) Cloning

isolation of a defined piece of DNA (usually containing a gene) and producing many copies of that sequence usually by putting in bacteria for further application

Cloning genes is possible because of 2 things:

1. the ability to cut and paste DNA

   • possible with discovery of restriction enzymes

2. the ability to isolate and amplify those cut and pasted DNAs

   • possible because DNA can be introduced into bacteria and replicated separate from the bacterial chromosome

Restriction Enzymes

part of the restriction/modification system of prokaryotes

• bacterial immune system

Modification Portion of System

chemical modification of bacterial DNA by methylation

Restriction Portion of System

enzymatic cutting of unmodified DNA rendering it non-functional

• restriction endonucleases

Methylated DNA

• small chemical modification to bases in the DNA

• does not affect base pairing

• but does affect protein interaction with the DNA

Types I and III Restriction Endonucleases

have both modification and restriction functions, not useful

Type II Restriction Endonucleocases

• have separate restriction and modification enzymes, very useful

• have sequence-specific recognition site

• reproducibly digest DNA into “bite-sized” pieces

• recognition sites are palindromic (read the

  same in either direction)

• many different enzymes available recognizing different sequences

• These “restriction” enzymes were first discovered by Smith and Wilcox in 1970 - pioneered the science of molecular biology

Restriction Mapping of Lambda Phage Chromosome

• If the purified chromosome is first digested with ApaI, two fragments, one measuring 10.1 kb and the other 38.4 kb, are generated, indicating that ApaI must cut the genome once

• If we digest the purified chromosome with XhoI, two fragments, one 33.5 kb and one 15 kb, are generated, indicating that XhoI must also cut the genome once

• To determine which order is correct, we need to perform a double digest, in which both enzymes are used simultaneously to cut the lambda genome.

• This experiment generates three pieces: 10.1 kb, 15 kb, and 23.4 kb. Since the 15-kb XhoI fragment remained intact but the 33.5-kb XhoI fragment was cut into two fragments (10.1 kb and 23.4 kb) by ApaI

• The other possible map can be eliminated as incorrect since it would generate fragments of 4.9, 10.1, and 33.5 kb

Restriction Enzymes

• always cut in the same place at the same sequence leaving the same ends

• and DNA is DNA is DNA

• Therefore, two different DNAs can be “spliced” together if cut with the same enzyme

• recombinant DNA

• Therefore, pieces of DNA can be “cut” from organism and “pasted” into plasmids

Plasmids

• naturally occurring molecules of DNA that occur in bacteria

• many of them contain genes for antibiotic resistance

• Circular DNA molecules that replicate autonomously in bacteria and usually carry nonessential genes

• Have been modified so that they can be used as cloning vectors

• Can be introduced into bacteria

  • bacterial transformation

• The F factor involved in E. coli conjugation

  • When used as cloning vectors replicate independently of the bacterial chromosome and, unlike the F factor, which can recombine into the E. coli chromosome, always remain separate from it.

  • most have been modified in the laboratory to possess several features that facilitate the production of recombinant DNA molecules

Cloning Vectors (Plasmids)

Contain:

1. restriction enzyme recognition sequences

   • (so DNA can be inserted)

2. a bacterial origin of replication

   • (so it can replicate in bacteria)

3. a selectable marker

   • (antibiotic resistance)

Making Recombinant DNA Molecules

• Although it is common to cut both source and vector DNA with the same enzyme, variations on this theme are frequently employed.

• For example, two different restriction enzymes that create complementary sticky ends are sometimes used.

• When different restriction enzymes are used to digest vector and donor DNA, complementary sticky ends are called cohesive compatible ends.

BamHI

• recognizes the 6-bp sequence

• and leaves sticky ends

• Sau3A recognizes the 4-bp sequence

• and leaves sticky ends

Directional Cloning of DNA Molecules

• A feature of experiments using a single restriction enzyme or using two enzymes with cohesive compatible ends is that the insert DNA can be ligated into the vector in either orientation.

• One way to ensure that DNA to be cloned is inserted into a vector in a specific orientation is to use two restriction enzymes that each cut a different sequence, thus creating two different sticky ends on the vector that are compatible with the same nonidentical sticky ends of the insert

Three Desirable Features of Directional Cloning

1. only insert-DNA fragments possessing the two different compatible ends will be efficiently inserted into the vector

2. the inserted fragments are ligated in a particular orientation dictated by the cohesive compatible ends

3. due to the incompatibility of the two ends of the digested vector DNA, the vector cannot re-ligate to itself, thus minimizing the creation of nonrecombinant

   vectors

Connecting blunt ends to create recombinant DNA molecules:

Some restriction enzymes naturally create blunt ends, but any restriction enzyme site can be converted into a blunt end

Amplification

replication of the recombinant DNA molecules in large numbers—the recombinant molecules are introduced into E. coli by transformation

• The process was described by Griffith Frederick, Oswald, Colin, and Maclyn in their early investigations of the hereditary function of DNA

How do you tell if cloning has been successful?

Antibiotic selection (tells you that the plasmid has been established in the cell)

How do you know that the plasmid is truly recombinant?

• if a cell survives on plates with antibiotics, it means that it has a plasmid

• but it does not mean that the plasmid has an insert

• this can be determined using a plasmid with a lacZ gene (= bacterial gene coding for beta-

  galactosidase)

• blue/white screening

• LacZ gene intact - blue colonies

• LacZ non-functional - white colonies

Dilute Salt Solution on Ice

solidifies membrane mask charges

Heat Shock

causes fluid influx causing uptake of DNA

Cells must be made “competent” to take up DNA:

1. dilute salt solution on ice solidifies membrane mask charges

2. heat shock causes fluid influx causing uptake of DNA

3. Once inside: DNA is repaired, replicated and antibiotic resistance expressed

   = transformation of bacterial cells

Genomic Libraries

• are collections of individual clones derived from the genomic DNA of an organism

• genomic DNA, usually from a single individual, is isolated and fragmented into smaller pieces that are then ligated into cloning vectors

• he recombinant vectors are transformed into bacteria that grow into colonies that collectively contain clones representing the entire genome

Construction of cDNA Libraries

• The starting material is mRNA, often derived from a specific tissue or cell type.

• Messenger RNA cannot be cloned directly because it is single-stranded and is of course RNA, not DNA.

• Cloning of mRNA sequences can be accomplished by synthesizing a double-stranded cDNA copy of the mRNA and then ligating the cDNA into a vector.

• These libraries are especially useful for working with eukaryotic organisms whose gene sequences are interrupted by many long introns.

Expression of Heterologous Genes in Bacterial Hosts

• Bacterial transformation by a recombinant plasmid is the primary method for generating transgenic bacteria.

• Expression vectors are vectors that have been furnished with sequences capable of directing efficient transcription and translation of transgenes

• Expression vectors for use in E. coli are constructed from plasmids that have been equipped with promoter sequences that bind RNA polymerase upstream of the multiple cloning site (MCS) of the plasmid.

• Efficient translation of mRNA in E. coli also requires the presence of a Shine–Dalgarno sequence in the untranslated region of the mRNA, another feature that is built into E. coli expression vectors.

Crown Gall Disease caused by Agrobacterium Via Plant Transformation

• The most widely used method of generating transgenic plants takes advantage of a natural plant transformation system that has evolved in the soil bacterium Agrobacterium tumefaciens.

• In nature, this bacterium is the cause of crown gall disease, an uncontrolled cell division in plant cells. This disease results in tumors (galls), typically at the crown (the base near the soil) of the plant.

• Wild strains of A. tumefaciens harbor a large plasmid (200 kb) called the tumor-inducing plasmid, or Ti plasmid

• A portion of the Ti plasmid, a region referred to as the transfer DNA (T-DNA) is transferred from the bacterium into the nucleus of a plant cell.

• Once inside the plant cell, the T-DNA can recombine illegitimately with the plant nuclear genome, resulting in an insertion of the T-DNA at a random location in the plant genome