In gene cloning, multiple copies of a gene are isolated. One or more genes to be cloned in a large complex genome are transferred to a small simple genome.

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BACTERIAL GENETICS AND GENE CLONING

 In gene cloning, multiple copies of a gene are isolated.

 One or more genes to be cloned in a large complex genome are transferred to a small simple

genome.

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BACTERIAL GENETICS AND GENE CLONING

 This process is also known as in vitro recombination

because it is carried out in vitro (in a test tube).

 Restriction enzymes, DNA

ligase, PCR and synthetic DNA

are used in molecular cloning

studies.

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Use of Restriction Enzymes to determine DNA base sequences

 DNA, which is fragmented from restriction enzymes by specific restriction enzymes, is separated by agarose gel electrophoresis to determine the size of these fragments.

 Restriction mapping of DNA can be performed using different restriction enzymes. Such

restriction assays are used to show differences between two or more DNAs.

 These DNA fragments in the agarose gel can then be removed from the gel and used for

hybridization or sequencing.

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 The DNA is separated into single strands by heating (denaturation).

 When the temperature is lowered, these single strands become double stranded again

(annealing).

 These single-stranded DNAs can be combined with single-stranded DNAs or RNAs from

different sources. This process is called

hybridization. Homologous regions in single strands obtained from different sources are determined.

 Hybridization is used in Southern Blot and

Northern Blot techniques.

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Determination of DNA base sequences

 Maxam-Gilbert method based on chemical breakdown of DNA

 Chemicals break down DNA from four regions: G, A + G, C and C + T.

 DNA fragments are separated by electrophoresis according to their size. It is determined by

exposure of the gel to x-rays (autoradiography).

 Sanger method: Dideoxynucleotides (containing

no 3'-OH group of deoxyribose) are added to the

ends of DNA synthesized by DNA polymerases.

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Gene cloning is carried out mainly in three stages

1. DNA isolation:

2. Binding of DNA fragments to a vector by DNA ligase:

Consolidation of DNA from different organisms and non-homologous sources (chimeric DNA)

3. Transferring the cloned DNA to the host:

Natural transfer mechanisms

Pathways developed under laboratory conditions

(protoplast fusion, protoplast transformation,

electroporation and microinjection)

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Cloning Vectors

 Should be suitable for in vitro studies

 Efficient replication in the cell

 Suitable restriction sites to which the DNA to be cloned can be inserted must be in the vector

 For these cloning sites on the vector, single- cut restriction endonuclease sites are very important. In this way, foreign DNA can be linked to a particular region of the vector.

 The vector should have a property (such as antibiotic resistance) that allows easy

identification of the recombinant molecule.

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Plasmids

 They have a small genome with supercoiled structure and can be easily isolated.

 They can replicate themselves in the cell they entered.

 They make a large number of copies in the cell.

 They carry genes that are easily identifiable,

such as antibiotic resistance genes.

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Plasmid pBR322 that can replicate in E. coli

 A suitable size for cloning (4361 bp).

 Easy to isolate.

 It can normally be replicated to produce 20-30 copies in an E. coli cell. If the protein synthesis in the cell is inhibited by chloramphenicol, they can reach 1000-3000 copies in a cell (40% of the genome).

 They can bind foreign DNA (up to a maximum of 10 kbp).

 They have cut zones recognized by many restriction

enzymes. Each enzyme can cut plasmid from a single cut site.

 Since tetracycline and ampicillin have resistance genes, they can be easily identified in their cell. The restriction enzymes contain the cut-off regions of the resistance genes.

 They can be easily transferred to the cell by natural or artificial transformation.

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Phages

In the lambda genome, the region between the J and N genes that is not necessary for infection of the phage is replaced by

foreign DNA.

The phage DNA is isolated and cut with a suitable restriction enzyme.

The DNA ligase and the foreign DNA fragment are linked between the two lambda fragments. DNA fragments up to 20 kbp in size can be ligated.

By adding the cell extract containing the phage head and tail

proteins to the medium, the recombinant DNA is packaged into the head to form phage particles.

E. coli is infected and phage clones are isolated from plaques.

The foreign DNA sequence carried by the recombinant phage is determined by nucleic acid hybridization techniques by DNA sequence analysis.

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Cosmids

 Plasmid vectors containing the cos region of lambda phage.

 This region is necessary for the packaging of DNA into Lamda virions.

 Cosmids can infect E. coli.

 Cosmids are used to clone 50 kbp DNAs.

 With the use of cosmids, DNA is stored in a phage particle instead of the plasmid.

Phages are more stable than plasmids. It

can protect the recombinant DNA for a

long time.

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 Bacterial Chromosome

 Microbial Genomics

 The genes of a cell or a virus are

all called genomes. The genome

name includes all of the genome

mapping sequence analysis and

comparison of genomes.

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 The first genome to be sequenced in 1976 was the RNA of the MS2 phage with 3569 nucleotides.

 A year later, in 1977, the first DNA genome sequence was determined in the single-

stranded DNA phage, the 5386 nucleotide Φ X174 phage.

 The first cellular genome sequence was determined in 1995 in the bacteria

Haemophilus influenzae with 1,830,137 bp chromosome.

 After 2000, sequence analysis of human

genome was performed.

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Proteomics

 Proteome is any protein present at any time in a cell, tissue, or organism.

 Proteomics (functional genomics), on

the other hand, is defined as the structural

and functional properties and regulation of

the proteins of an organism to a large extent

depending on the genome.

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