Download Cloning :-

Biotechnology & Genetic Engineering Lecture ( 10 ) Assi. Prof. Rajaa Al –Anbaqi
Cloning :What is Cloning ?
The terms recombinant DNA technology ,DNA cloning ,molecular cloning ,or
gene cloning all refer to the same process ; the transfer of a DNA fragment of
interest from one organism to a self –replicating genetic element such as a
bacterial plasmid ( cloning vector ) . The DNA of interest can then be propagated
in a foreign host cell . This technology has been around since the 1970 s , and it has
become a common practice in molecular biology labs today . Cloning is frequently
employed to amplify DNA fragmentes containing genes , an essential step in their
subsequent analysis .
How does molecular cloning work ?
Cloning of any DNA sequence involves the introduction of a foreign piece of DNA into
an extra chromosomal element ( cloning vector ) of an organism which then produces
copies of the vector as it replicates itself ,thereby amplifying the DNA of interest .
The whole process can be summarized in the following steps : fragmentation , ligation
,transfection , screening / selection , and conformation of ins
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The first recombinant DNA molecules were generated at Stanford University in 1972,
utilising the cleavage properties of restriction enzymes (scissors) and the ability of DNA
ligase to join DNA strands together (glue). The importance of these first tentative
experiments cannot be overestimated. Scientists could now join different DNA
molecules together and could link the DNA of one organism to that of a completely
different organism. The methodology was extended in 1973 by joining
DNA fragments to the plasmid pSC101,which is an extra chromosomal element
isolated from the bacterium Escherichia coli. These recombinant molecules behaved as
replicons; that is, they could replicate when introduced into E. coli cells (host cells).
Thus, by creating recombinant molecules in vitro, and placing the construct in a
bacterial cell where it could replicate in vivo, specific fragments of DNA could be
isolated from bacterial colonies that formed clones (colonies formed from a single
cell, in which all cells are identical) when grown on agar plates. This development
marked the emergence of the technology that became known as gene cloning (Fig. 1.3)
Or another way for explain it: The mainstay of genetic manipulation is the ability to
isolate aGene cloning enables isolation and identification of individual genes. Single
DNA sequence from the genome. This is the essence of gene cloning and can be
considered as a series of four steps (Fig. 1.1) A useful analogy is to consider gene
cloning as a form of molecular agriculture, enabling the production of large amounts (in
genetic engineering this means micrograms or milligrams) of a particular this ability to
isolate a particular gene sequence is still a major aspect of gene manipulation.
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1- The first step requires basic step it generation of DNA fragments (see map1)
by
Preparation of DNA fragments for sequencing is largely dependent on the scale of the
sequencing project. If the aim is to sequence part of a gene that has already been isolated
and identified, the process is relatively straightforward and usually requires that the
fragment is of a suitable length and in a suitable form for the sequencing procedure in use
If, however, the aim is to sequence a much larger piece of DNA (such as an entire
chromosome), the problem is much greater. In this case the sequencing strategy is
important, and there are two approaches to this. The first is an ordered sequencing
strategy. The second is the so-called shotgun sequencing method, where the fragments
are generated and processed at random. Assembly of the sequence is then carried out by
searching for sequence overlaps using a computer. The two strategies are shown in 3.7
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Generation and preparation of DNA fragments is fairly simple on a purely technical
level. The fragments are often cloned sequences that are presented for sequencing in a
suitable vector; with careful attention to detail where the fragment is within the genome;
there are basically two approaches. The first is an ordered sequencing strategy. The
second is the so-called shotgun sequencing method , The sequencing protocol is
essentially a technical procedure. There are two main methods for sequencing DNA.
The first is a chemicals and the second is Enzymatic method . but the most widely used
techniques are based on the enzymatic method .Whatever the method , the desired result
is to generate a set of overlapping fragments that terminate at different bases and differ in
length by one nucleotide. This is known as a set of nested fragments. The final stage
Analysis of fragments is similar for both methods (chemicals and enzymatic methods))
it usually involves separation of the fragments on a polyacrylamide gel (electrophoresis)
and autoradiography which are dentified as they pass a detector. When the generation of
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fragments (for enzymatic sequencing or chemical sequencing ) and usually involves
subcloning into different vectors.
2- Joining to a vector or carrier molecule
Gene cloning is achieved by using a vector (carrier) to propagate the desired sequence
in a host cell. Choosing the right vector/host combination is one of the critical stages of a
cloning procedur. Many plasmid vectors are now available and some types can be used
directly for DNA sequencing experiments. Another method is to clone the DNA into a
vector such as the bacteriophage M13, which produces single-stranded DNA during
infection. This provides a suitable substrate for the sequencing reaction .The various
types of host cell and vector systems and methods for getting DNA into cell .We need
now focus on A- the vectors characters , B-types of vector systems and C- methods
for getting DNA into cell .
A- There are certain essential features that vectors must posses:-Ideally
1- They should be fairly small DNA molecules, to facilitate isolation and handling.
2- There must be an origin of replication, so that their DNA can be copied and thus
maintained in the cell population as the host organism grows and divides.
3 - It is desirable to have some sort of selectable marker that will enable the vector to be
detected.
4- The vector must have at least one unique restriction endonuclease recognition site to
enable DNA to be inserted during the production of recombinants.
Plasmids have these features and are extensively used as vectors in cloning experiments.
B- Types of vector systems:- There are two main types :1- Plasmids :- Many types of plasmids are found in nature, in bacteria and some yeasts.
They are circular DNA molecules, relatively small when compared to the host cell
chromosome and have been Specially constructed. Plasmids are extrachromosomal
genetic elements that are not essential for bacteria to survive but often confer
advantageous traits (such as antibiotic resistance) on the host cell. The antibiotic
resistance genes encoded by plasmid DNA (pDNA) are often used in the construction of
vectors for genetic engineering, as they provide a convenient means of selecting cells
containing the plasmid. When plated on growth medium that contains the appropriate
antibiotic, only the plasmid-containing cells will survive. This is a very simple and
powerful selection method asmid found in the host cell, known as the copy number. ALow-copy-number plasmids . B- High copy- number plasmids
2- Phages :-
The term ‘bacteriophage’ is often shortened to ‘phage’ and can be used to describe
either one or many particles of the same type. Thus, we might say that a test tube
contained one ^ phage or 2 × 106 _ phage particles. The plural term ‘phages’ is used
when different types of phage are being considered; we therefore talk of T4, M13, and
^as being phages Bacteriophages are essentially bacterial viruses and usually consist of a
DNA genome enclosed in a protein head (capsid). As with other viruses, they depend on
the host cell for their propagation and do not exist as free-living organisms. Structurally,
phages fall into three main groups: (1) tailless (2) Bacteriophages are essentially bacterial
viruses and usually consist of a DNA genome enclosed in a protein head (capsid). As
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with other viruses, they depend on the host cell for their propagation and do not exist as
free-living organisms. head with tail, and (3) filamentous. The genetic material may be
single or double-stranded DNA or RNA, with double-stranded DNA (dsDNA) found
most often. In tailless and tailed phages the genome is encapsulated in an icosahedral
protein shell called a capsid (sometimes known as a phage coat or head).
The genome of phage ^ is 48.5 kb in length, and encodes some 46 genes (Fig. 5.4). The
entire genome has been sequenced (this was the first major sequencing project to be
completed, and represents one of the milestones of molecular genetics), and all the
regulatory sites are known. At the ends of the linear genome there are short (12 bp)
single-stranded regions that are complementary. These act as cohesive or ‘sticky ends,
which enable circularisation of the genome following infection. The region of the
genome that is generated by the association of the cohesive ends is known as the cos site
. The utility of phage ^ as a cloning vector depends on the fact that not all of the ^
genome is essential for the phage to function. Thus, there is scope for the introduction
of exogenous DNA, although certain requirements have had to be met during the
development of cloning vectors based on phage^ . First, the arrangement of genes on the
^ genome will determine which parts can be removed or replaced for the addition of
exogenous DNA. It is fortunate that the central region of the ^ genome (between
positions 20 and 35 on the map shown in Fig. 5.4) is largely dispensable, so no complex
rearrangement of the genome in vitro is required. The central region controls mainly the
lysogenic properties of the phage, and much of this region can be deleted without
impairing the functions required for the lytic infection cycle. Second, wild-type^ phage
will generally have multiple recognition sites for the restriction enzymes commonly
used in cloning procedures. This can be a major problem, as it limits the choice of sites
for the insertion of DNA. In practice, it is relatively easy to select for phage that have
reduced numbers of sites for particular restriction enzymes, and the technique of
mutagenesis in vitro may be used to modify remaining sites that are not required. Thus, it
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is possible to construct phage that have the desired combination of restriction enzyme
recognition sites.
Life cycle of bacteriophago .
3- Other vectors :- Cosmids and phagemids incorporate some of the characteristics of
both plasmid and phage-based vectors. Cosmids , This has been exploited in the
construction of vectors that are made up of plasmid sequences joined to the cos sites of
phage^.Hybrid plasmid/phage vectors in which the phage functions are expressed and
utilised in some way are known as Phagemids .
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3-introduction into a host cell for amplification :- back to map 5
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