Summarize the following text to main points and sub-points and make detailed explain for them:

 A 
gene library is formed and the required cloned gene is 
selected. 
2 - The use of processed (spliced) mRNA transcribed from 
the required gene, as a template for the synthesis of the DNA 
of that gene by the enzymatic action of reverse transcriptase. 
The resulting DNA is usually called c-DNA. This approach is 
particularly useful in solving the problem of cloning an 
eucaryotic gene containing exon sequences and the absence 
of RNA splicing enzymes in procaryotic organisms . 
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3 - SYNTHETIC DNA: An approach known as reverse 
translation is used to deduce the sequence of DNA segments 
from the sequence of amino acids in corresponding proteins. 
First, nucleotide sequence of corresponding mRNA is 
deduced from the amino acid sequence of protein. After 
determining the mRNA sequence, the complementary DNA 
sequence can be deduced and synthesized in vitro. 
Degeneracy could complicate this method. Synthetic DNA's 
which code for up to 200 amino acid long proteins could be 
synthesized artificially. 
This approach has been used for the production of 
recombinant human insulin in bacteria. 
2- ENZYMES: 
1- Suitable endonuclease. 2- Ligase. 
3- CLONING VECTOR: 
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The genetic element, which can be used as a vector, must 
meet the following requirements: 
1- It must be capable of autonomous replication, i.e. 
independent of main chromosome. This facilitaes the 
amplification and isolation of the vector carrying the gene. 
2- It must has enough non-essential DNA segments which 
can be broken for the insertion of the foreign gene, without 
affecting the ability of the vector to replicate. 
3- A large amount of foreign DNA can be inserted. 
4- It must carry genes (marker genes) that help in screening 
and/or selection of the cloned host. This type of genes could 
confer a new property (phenotype), on the cloned host, so as 
it helps differentiate the cells that received the vector from 
cells which did not; e.g. the acquisition of ability to ferment a 
certain sugar. 
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3- CLONING VECTOR: 
The genetic element, which can be used as a vector, must 
meet the following requirements: 
1- It must be capable of autonomous replication, i.e. 
independent of main chromosome. This facilities the 
amplification and isolation of the vector carrying the gene. 
2- It must has enough non-essential DNA segments which 
can be broken for the insertion of the foreign gene, without 
affecting the ability of the vector to replicate. 
3- A large amount of foreign DNA can be inserted. 
4- It must carry genes (marker genes) that help in screening 
and/or selection of the cloned host. This type of genes could 
confer a new property (phenotype), on the cloned host, so as 
it helps differentiate the cells that received the vector from 
cells which did not; e.g. the acquisition of ability to ferment a 
certain sugar. 
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Selection of cloned host cells could be also made 
simpler by the presence of an antibiotic-
resistance gene. 
The growth of host cells in that particular 
antibiotic would be possible only when it carry 
such selection gene. 
5- It must have a single restriction site for at least 
one endonuclease type. 
6- Must not be able to move from one cell to 
another. 
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7- Undergo amplification by chloramphenicol 
treatment. 
Host cells can accommodate much more copies of 
small plasmids than large ones. Chloramphenicol 
(an inhibitor of bacterial protein synthesis) 
inhibits the initiation of replication of the main 
chromosome but not the plasmid. 
Chloramphenicol also inhibits the synthesis of 
regulatory repressor protein responsible for 
regulation of plasmid replication. As a result, the 
copy number of plasmid could reach 1000 copies 
per cell or more. 
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8- It must be stable inside the host cell. 
Types of cloning vectors: 
Plasmids: Small to medium size plasmids 
are most suitable because they can be 
amplified by chloramphenicol, they are 
non-conjugative so cannot move from one 
cell to another, and they are easily 
introduced to host cells by transformation. 
Phages: Many phages can be used as cloning 
vectors. 
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Especially important in that respect are some temperate 
phages such as lambda phage, and filamentous phages 
such as M13. Modified lambda phages but not wild types 
are suitable as vectors. Wild-type has more than a single 
restriction site on its genome. The modified phage was 
treated so as to remove unwanted restriction sites. 
Lambda phage vector cannot accommodate much 
foreign DNA because of restriction imposed by the 
capsid space. Also the virus shows considerable 
amplification only during the lytic cycle of 
multiplication. 
Animal viruses: For cloning mammalian cells, viruses 
such as SV 40 and retro-viruses have been used for 
cloning human tissue cell-lines 
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EXPRESSION VECTORS: 
An expression vector is one which contains, in addition to the 
cloned gene, all regulatory sequences required for the 
expression of the cloned gene. Elements of gene expression 
are those involved in transcription and translation. 
Expression vector is constructed so that it has the following 
properties: 
1 - Can achieve high copy number. The higher the copy 
number, the better the expression of the gene. 
2- Has high efficiency promotor site. 
3 - The promotor must be well regulated e.g. lac or trp 
promotors. 
4- Its mRNA must have the proper ribosome-binding site.
 5- Has the proper open reading frame. 
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4- MICROBIAL HOSTS FOR THE CLONING VECTOR: 
A suitable host for cloning vectors better has the following 
characteristics: 
1- Well characterized genetically and biochemically. 
2- Rapid grower in cheap media. 
3- Not pathogenic. 
4- Transformable by DNA. 
5- Free from harmful restriction endnuclease activity to 
avoid degradation of cloned gene. 
Microorganisms most commonly used for cloning are 
Escherichia coli, Bacillus subtilis and Saccharomyces 
cerevisiae. Cloning in mammalian host cells was also 
achieved cell cultures. 
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Escherichia coli have few disadvantage including their 
content of endotoxin, and it tendency to colonize the 
human intestine and its potential pathogenicity. 
Bacillus subtilis does not produce endotoxin and is non 
pathogenic. 
The main problem with this bacterium is plasmid and 
foreign gene instabilities, and loss of plasmid's ability to 
replicate upon repeated subcultures. 
5- SUITABLE SCREENING METHOD FOR CLONED GENE: 
The right clone could be screened for with high degree of 
selectivity by looking for: 
1) The expressed recombinant protein in question, or 
2) The cloned gene itself (when protein is not expressed). 
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Screening for recombinant protein: Screening 
method depends on the type and/or function of 
the protein. 
Enzymatic activity: If the recombinant protein 
was an enzyme, colonies producing this enzyme 
can be detected biochemically, e.g. by the 
inclusion of a substrate for the enzyme which in 
the growth culture medium; the product could 
either be colored or can be detected by color-
indicators. Colored colonies are then picked and 
propagated in pure culture. 
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Immuno-assays: Radiolabeled or enzyme-labeled antibodies 
raised against the protein could be used to detect the 
presence of recombinant protein in partially lysed colonies or 
in culture filtrates. Radioactivity or development of color 
upon addition of substrate would indicate the presence of the 
protein in question. 
GENERAL METHOD FOR GENE CLONING: 
When two different DNA's e.g. a human DNA and a plasmid, 
are to be joined together, they must have staggered (sticky) 
ends that are rightly complementary so that they join 
together. 
Both DNA's must have the same palindrome and are treated 
with the same endonuclease that nicks these palindromes, so 
that complementary ends join easily. As mentioned before, 
the plasmid (vector) should have a single palindrome site of 
each type. 
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The same enzyme is used to cleave the human DNA. 
Because of complementary sticky ends, when cleaved 
plasmid and human DNA are mixed together, they join 
together and a circular plasmid carrying a human DNA 
fragment is thus generated. 
Increasing numbers of pharmaceutical products of great 
medical benefit are being developed and marketed. 
Some important examples of recombinant proteins are 
presented in table. 
The recombinant DNA is introduced into the proper 
microbial host by transformation. The transformed 
recombinant gene undergoes amplification in the host 
and as a result of multiplication of the host. 
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Colonies carrying various cloned genes are replicated by 
replica platting. Colonies are then subjected to lysis to 
release their DNA contents in situ, which are denatured 
in order to obtain single strand DNA (preparation for 
hybridization). 
Colonial DNA is transferred to nitrocellulose filters 
where they bind strongly. 
DNA's of colonies are then treated with a gene probe 
having complementary sequence to the searched for 
gene. 


Summary :