A detail description about vectors and its types and comparison between different types of vectors

1. Comparison Between
Different Types Of
Vectors
Presented by Fahima Ashraf Kasi

2. Content
 Introduction
•Cloning vector
•Why cloning vector
•Features of C.V
•Choice of C.V
Different cloning vector its
advantages and disadvantages
 plasmid cloning vector
pBR322
Bacteriophage lambda cloning vector
 pUC19
Cosmid
YEp13

3. CLONING VECTORWhy cloning vector

4. CLONING VECTOR
 Small piece of DNA
Taken virus, plasmid or the cell of
a higher organism that can be
maintained stably in an organism
into which a foreign DNA fragment
can be inserted for cloning
purpose
 Contain features 
insertion or removal of
DNA fragments  in or
out
 Most vectors are genetically
engineered. Choose
according to size and type of
DNA to be cloned .
 After DNA has been cloned
into a cloning vector  sub
cloned into another vector
design for specific use

5. Cloning vector
The molecular analysis of DNA has been made possible by
the cloning of DNA. The two molecules require for cloning
are .
 DNA to be
cloned
 Cloning
vector

6. Why Cloning
Vector?
1) Amplify a single molecule
of DNA into many copies
4) Without cloning vector, Molecular
gene cloning is totally impossible.
2) Vehicle to artificially carry
foreign genetic particles into
another cell, where it can be
replicated and expressed
3) Transport the cloned sequence
between biological host and test
tube
.

7. Features of cloning vector
All commonly used cloning vectors have some essential features
 Origin Of replication (ori)
• This make autonomous replication in a vector.
• Ori is a specific sequence of nucleotide from where replication
start
• When foreign DNA is linked to the sequence along with vector
replication, foreign(desirable) also start replication within host
cell.
 Cloning site/ Restriction site
 A place where vector DNA can be digested and desired DNA
can be inserted by the same restriction enzyme
 Point of entry or analysis for genetically engineered work
 Most plasmid contain a multiple cloning site (MCS) which
have many (up to -20) restriction sites

8. Features of cloning vector
 Selectable markers
• These are the genes that confers resistance to particular
antibiotics or selective agents that would normaly kill the
host cell or prevent its growth
• A cloning vector contain selectable markers, which confer
on the host an ability to survive and proliferate in a selective
growth medium containing tbe particular antibiotics
 Reporter gene
• Used in cloning vector to facilitate the screening of
successful clones by using features of these genes that
allow successful clone to be easily identified
• Such features are used in blue/white selection

9. What determines choice of vector?
vector Insert size Vector size Organism
pBR322 <10kb 4361 kb E.coli
Bacteriopha
ge
9-15kb 48.5kb E.coli
Cosmid 23-45kb 52kb E.coli
• Insert size
• Vector size
• Restriction size
• Cloning efficiency
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10. Self replicating, double
stranded, circular DNA
molecules, independent
extra chromosomal entities .
Range in size  less
than 1 to more than
500kb.
Can take GOI of up to
10kb in size
Population of plasmid in a
bacterium 0.1-5.0% of the
total DNA.
Example: pBR322,F
plasmid ,pUC19 etc.
Plasmid cloning vector

11. Plasmid cloning vector
Disadvantages
Cannot accept large
fragments
Size range from 0-10kb
Standard method of
transformation is insufficient
Advantages
Replication independent of host
cell
Several copies may be present
Have antibiotic resistance (easy
detection )

12. pBR322
The Nomenclature of pBR322
 In pBR322 the “p” stands for plasmid
 BR recognizes the work of researchers
B: F. Bolivar
R: R.Rodriguez
 322 is used to distinguished it from other
plasmid (pBR325, pBR327 etc.) developed
in the same labortary

13. The construction of pBR322
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Size :
pBR322 contain 4,361bp
.
Origin of replication
Selectable markers
It carries two antibiotic resistance genes—ampicillin and tetracycline
It carries a fragment of plasmid pMB1 that acts as an origin for DNA replication and thus
ensures multiplication of the vector
Cloning sites
It carries a number of unique restriction sites. Some of these are located in one of the
antibiotic resistance genes (e.g., sites for Pst I, Pvu I, and Sac I are found in Amp and
BamHI and Hind III in Tetr).

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15. pBR322
It has very high mobility. Due
to this, the vector may get
lost in a population of mixed
host cells.
01
There is a limitation in the size of
the gene of interest that it can
accommodate.
02
The screening process is time-
consuming and laborious.
03
Small size (~ 4.4 kb) enables easy
purification and manipulation 01
Two selectable markers (Amp
and Tet) allow easy selection of
recombinant DNA.
02
It can be amplified up to
1000-3000 copies per cell
03
Advantages
Disadvantages

16. Bacteriophage lambda vector
 Phage lambda is a bacteriophage or phage i.e. bacterial virus that use E.coli as a
host
 It structure is similar to a typical phage head tail and tail fibers
 The lambda viral genome is 48.5kb with a 12 base ssDNA “sticky end” at the both ends
These ends are complementary to each other and can hybridize to each other forming
cohesive end (cos site)

17. Bacteriophage lambda vector
 Infection: lambda tail fibers absorbed through cell surface receptors, tail
contract, DNA is injected. The DNA is circularized and lambda begins its life
cycle in E.Coli
 There are two kinds of lambda phage vector : insertion vector and
replacement vector .
 Insertion vector contain unique cleavage site where foreign DNA with size 5-
15 kb may be inserted
 Replacement vector : here they cleave the region that are not necessary for
lytic cycle. They might be deleted and replaced with foreign DNA (gene of
interest) in cloning purpose

18. Genetically engineered lambda phage cloning vector
• Bacteriophage lambda cloning vector that have been devised has
two BamHI site that flank the I/E region.
• When purified DNA from this bacteriophage is cut with BamHI, 3
segments are created
1. Left arm (L region): contain genetic information for the production
of head and tail
2. Right arm ( R region): contain gene for replication and cell lysis
3. Middle I/E fragment : contain the genes for insertion and
replacement process

20. Objective
The objective of this protocol is to replace the middle segment of the
lambda DNA with the cloned DNA that is approximately 20kb in length
uses
The main use of all lambda based vectors is to clone DNA fragments that
are too long to be handled by other plasmid

21. Advantages and Disadvantages of lambda phage vector
It is frequently quite difficult to
isolate large quantities of DNA
3.Transfection of E. coli is much
easier with phage particles.
Less easy to handle1. Storage of phage particles is
comparatively much easier than
that of plasmid based vectors.
2.The shelf-life of phage particles is infinite.
Advantages Disadvantages
There is still no truly rapid, reliable
protocol for the production of very
clean lambda-DNA.

22.  pUC19 is one of a series of plasmid cloning vectors
created by Joachim Messing and co-workers.
 Nomenclature
 The designation "pUC" is derived from the classical
"p" prefix (denoting “plasmid") and the abbreviation
for the University of California, where early work on
the plasmid series had been conducted.
What is pUC19?

23. .
1. origin of replication
2. selectable markers
3. lac z gene having MCS
Structure of pUC19

24. Characteristics of PUC19
• pUC19 is a small, high copy cloning vector for replication in E. coli.
• Contains2868 bps.
• Puc19 are obtained by modifying the pbr322 vector.
• These are smaller then pbr322 of being only approximately 2.7kb.
• It produce 500-600 copies.
• It has been constructed using the ampicillin resistance gene and the pMB1 origin of
replication from Pbr322.
• The pMB1 of pUC19 differs from the pBR322 origin by a single point mutation and the
lack of the rop gene, leading to a high copy number.
• Uses
• Cab be used both as
Cloning vector
Expression vector
Characteristics of pUC19

25. 1. Produces of high copy
no of 500-600 copies
per cell
It cannot accommodate a gene of
interest larger than 15kb
2. Easy and single step
selection.
Disadvantages
Advantages
Advantages and
disadvantages of pUC19

26. .
• It is the most sophisticated type of lambda based vector.
• Can carry 40 kb of cloned DNA and can be maintained as
plasmids in e.coli.
•
A cosmid is a plasmid that contains phage sequences that allow
the vector to be packaged and transmitted to bacteria like a phage
vector
• Cosmid are hybrid between phage DNA molecule and vector
plasmid.
Cosmid vector

27. .
• Basically a plasmid that carries a cos site.
• Origin of replication.
• Contain selectable markers e.g amp resistant gene.
Structure of cosmid

28. Uses
• Used for the construction of genomic libraries of eukaryotes.
Uses

29. .
• Used to clone gene of interest
up to 40 kb.
• As the lambda phage will insert
the recombinant DNA into the
host cell, an extra step of
inserting the recombinant DNA
into the host cell is not
performed.
• Cannot accept larger
fragments.
Advantages & disadvantages

30. .
• A vector, usually a plasmid constructed so that it can propagate in
two different host species.
• Why called as the shuttle vectors?
• Since these vectors can be grown in one host and then moved into
another without any extra manipulation.
Types
1. Eukaryotic-prokaryotic shuttle vectors.
2. Prokaryotic-prokaryotic shuttle vectors
Shuttle vectors

31. Continue…
• These vectors have been designed to replicate in cells of two
different species, therefore they contain two origins of replication .
• Created by recombinant DNA techniques.
• Example:yEp13 (yeast episomal plasmid)is an example of shuttle
vectors.
Continue…..

32. • ADVANTAGES
• They are capable of replicating into two or more types of hosts
including prokaryotic and eukaryotic cells.
• They replicate autonomously, or integrate into host genome and
replicate when the host cell multiplies.
• DISADVANTAGES
• The presence of two replication origins sometimes poses special
problems, one portion of replication origin of one species is totally
unrelated to another and interferes with the replication of other host.
Hence, in a shuttle vector various types of replication origins are to
be inserted and checked before experimenting.
Advantages and disadvantages

33. YEp13
• It illustrates several general features of east clonning vectors.
• Contains 2um origin of replication and selectable LEU2 gene ,also
includes the entire pBR322 sequence and therefore can replicated
and selected both in yeast an E.coli.
YEp13

34. Structure

35. Thank You.