1. Genomes Chapter 2: Studying DNA Terry Brown Third Edition
Copyright © Garland Science 2007

2. Chapter Objectives
Understand PCR and its application in genomics as well as PCR based cloning.
Clearly understanding of various enzymes and their function for genomic research.
Key features of cloning vectors.
Relationship of vector-host system and their application in genome library construction.

3. Tool Kit to study Genomics
DNA is invisible to naked eye
To manipulate DNA as desired way we need certain tools which can be effectively utilized to study genomes
The research during 1970, and 1980s resulted in the development of assembly of tools which can be utilized to work with DNA
Researcher identified and purified different enzymes which can:
Cut DNA
Ligate DNA
Copy DNA

4. This ability gave birth to recombinant DNA technology where DNA from different sources can be combined in a way that never existed before!
Recombinant DNA technology led to the development of DNA/Gene cloning where a piece or gene of interest can be manipulated in virtually any host
Making it possible to know the function of a gene in a model host

5. Enzymes for DNA manipulation
DNA polymerases
Nucleases
Ligases
End-Modification enzymes

6. DNA polymerases Replicate the genome Template-dependent DNA polymerase
Need primers
3’-5’ Exonuclease activity (proof reading)
5’-3’ Exonuclease activity (Primer removal)
Figure 2.2 Genomes 3 (© Garland Science 2007)

8. DNA polymerases in Research
First DNA pol I identified in E.coli by Kornberg and named as Kornberg Polymerase (contains both exonuclease activities)
A modified version of Kornberg Pol is called as Klenow Pol which lacks 5’-3’ exonucleases activity used for sequencing initially.
Thermostable DNA polymerases Taq Pol
Sequenase for Sequencing

9. Sequenase is used for sequencing due to its properties like:
High processivity
Negligible or zero 5’-3’ activity
Negligible or zero 3’5’ activtiy
RNA dependent DNA polymerases:
Synthesize cDNAs

10. Nucleases
The nucleases are very important and has DNA or RNA cutting ability
They fall into two classes
Exonucleases
Endonucleases
restriction endonucleses

11. Restriction endonucleases Enable DNA molecules to be cut at defined positions
There are three types of restriction endonucleses
Type I and III: (bind at specific sites but cut at different sites)
Type II: bind and cuts at specific sites
Produces blunt or flush ends
Produces sitcky or cohesive ends

12. Figure 2.5 Genomes 3 (© Garland Science 2007)

14. Examining the results of a restriction digest
The results of Restriction digestions are analyzed by
Agarose gel electrophoresis
Southern hybridization

15. Southern hybridization
Figure 2.6b Genomes 3 (© Garland Science 2007)

16. DNA Ligases
Two DNA fragments can be joined together by the help of DNA ligases which join them by introducing phophodiester bond.
Utilize ATP
NAD
Most widely used DNA ligase is from E.coli infected with T4 phage
Involve in T4 replication
The efficiency of ligation depends on
Concentration of DNA
The probability of two ends to come closer to each other so that ligase can work

17. The efficiency can be increased by the utilization of:
Linkers
Are short fragment of dsDNA containing a site for restriction endonuclease
Which will produce sticky ends after its digestion to facilitate cloning
Adoptors
Short fragment of dsDNA which have one end blunt and other end is already sticky so no need of digestion
Homopolymer tailing by terminal deoxynucleotidyle
Adds non template nucleotides on the 3’ end of DNA
Any polynuclotides can be added like poly G to facilitate cloning with poly C

18. End Modification Enzymes
Terminal Deoxynucleotidyl Transferase
Obtained from calf thymus tissue
Used for homopolymer tailing
Alkaline phophatase
Obtained from calf thymus tissue and E.coli
Removes phophate groups from the 5’ end of DNA
This prevents self ligation of the molecules
T4 polynucleotide kinase
Obtained from T4 infected E.coli
Performs reverse action of alkaline phophatase
It adds phophates to 5’-ends
The judicious use of alkaline phophatase and T4 polynucleotide kinase direct the action of DNA ligase in a predictive way

19. DNA Isolation
Chemical purification
Ion exchange purification of DNA

20. The Polymerase Chain Reaction (PCR)
PCR is a very powerful technique
Its impact on our understanding of genes and genomes has been immeasurable
PCR complements DNA cloning
Helps in purification of a segment of DNA by its amplification
The cloning works without knowing the sequence of DNA
While PCR has limitation that for utilizing PCR one need to know the flanking region of target DNA

21. Carrying out a PCR
The enzyme: thermostable DNA polymerase from Thermus aquaticus
Reaction mixture:
Buffer to maintain pH, salt and composition, medium to dissolve DNA and enzyme
Enzyme
Primers
Template
MgCl2
dNTPs
water
Conditions:
Denatureing
Annealing
Extension

23. DNA Cloning A process to ligate the DNA in a specific background
To have identical copies
To place the DNA in a new genetic background
To change the already present gene
The technique was developed due to the ability to:
Cut the DNA
Ligate it in some other place
The cloning was first invented in early 1970s and has revolutionized the field of molecular biology

24. Cloning Vectors The plasmids act as cloning vectors
They contains some genetic characters with provides some advantages like:
Origin of Replication
Selectable Marker
Screening Marker

25. Vectors Based on E. Coli The simplest vectors
Can be introduced into E. coli by the process of transformation
The most popular plasmid vector is pUC18 introduced in 1980s.
2.7 kb
Origin of replication
Two important genes
Selectable marker (ampicillin resistance gene) which produce β-latamase enzyme which breakdown the ampicillin
Secreening marker (LacZ’ segment of LacZ gene which produce β-galactosidase).
The lacZ’ produces α-peptide portion of β-galactosidase enzyme
This vector need to transformed in those E.coli strains which have modified LacZ gene and only can produce β-galactosidase when LacZ’ is present on plasmid enabling screening based on insertional inactivation
Develop blue color in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside).

26. Limitations
Can only accommodate small sized DNA like 10 kb at max
Larger segments interfere with plasmid replication.
Some sequences which are complementary to each other making secondary structure are not well handled by some strains of E. coli

27. Cloning vectors based on E. Coli bacteriophage genome
Bacteriophage are viruses which infects E. coli and replicate in them.
They are have two different type of mode of infection cycles of bacteriophage λ
Lytic infection cycle
Infect cell, multiply and breakdown the cell to release out
Lysogenic infection cycle
Infect cell, integrate in the bacterial genomic DNA, multiplicate as E. coli do, in some cell starts lytic cycle after excision from host DNA

28. The bacteriophage λ vectors can accommodate more DNA like 15 kb
The size of λ genome is 48.5 kb
15 kb segment contains genes needed for integration of phage DNA into host genome.
This portion which is “optional” can be deleted from its genome without impairing phage ability to replicate and synthesize new phage particles.
This portion can be utilized for cloning foreign DNA fragments
The linear genome of λ genome contains 12 nucleotides single stranded overhangs, called cos sites, which have complementry sequences and can base pair each others
Insertional vectors
In which optional region has been removed an a unique restriction site introduced at some position for cloning
Replacement vectors
In which optional regions remains within a stuffer fragment , which is flanked by a pair of restriction sites for replacement with desired fragment.

29. The cloning in λ genome can be done in two ways
Recircularization of genome by the help of cos sites and reintroduce it to E. coli by transfection
By in vitro packaging system which its cutting into tow arms i.e. left arm and right arm
Then the DNA is ligated with these arms
The successful clone will contains left and right arm at the outer side of cloned fragment
Only those DNA can be packed which have
Size of kb
Containing cos sites at both sides
The in vitro packaged viruses are used to infect E. coli which ultimately forms plaque from where be further analyzed for the presence of DNA
Blue white screening system can also be used with it.

30. Vectors for Longer Pieces of DNA
In λ phage particles 18 kb fragment of DNA can be cloned
Its higher than plasmid based vector but still very small compared with the sizes of intact genomes.
Smaller the cloning size larger the number of clones that would be required to make a genomic library

31. Cosmid Vectors Cosmid is a plasmid carrying λ cos site as the end.
Concatamers of cosmid molecules, linked at their cos sites, act as substrates for in vitro packaging.
So the particles containing cosmid DNA are infective but can not make new phage particles
But comsid can replicate due to the presence of ori in side the cell as plasmid.
About 44 kb of DNA fragment can be cloned into cosmids

32. Yeast Artificial Chromosomes
The development of YACs lead to the major breakthrough for cloning DNA fragments having more than 50 kb size.
These vectors are propagated in yeast i.e. saccharomyces cerevisiae
YACs are based on chromosomes rather than plasmids or virueses.
YACs contains three important components
The centromere (role in cell division)
Telomeres (important for protection of the ends)
Origins of replication (one or more for initiation of replication)

33. All these functionally important regions make about kb DNA, while yeast chromosomes range in size from 230 to 1700 kb.
There is a potential to clone Mb size DNA fragments in YAC
The standard YACs 600 kb DNA
But there are associated some problem of DNA instability

34. Other vectors Bacterial artificial chromosomes
Based on F plasmid of e.coli
Higher capacity of larger DNA
Lac selection
300 kb can be cloned in them
Very famous and been used in Human Genome Project
Bacteriophagte P1 Vectors
Very similar to lambda vectors, but P1 phage has larger genome
Can clone 125 kb DNA

35. P1-derived artificial chromosomes (PAC)
Combine features of P1 and BACs
Can clone 300 kb
Fosmids
Contains the F plasmid origin of replication and cos sites
Less prone to instability problems

36. Cloning in organisms other than E. coli
Cloning is not only useful for DNA coping rather it is used for expression analysis
For understanding the regulation
For producing things in other organisms like insulin
Vectors are developed for yeast and fungus
Based on 2 um circle a yeast plasmid
I.e. YIp5 vector (yeast integrative plasmid)
Its shuttle vector can replicate in e.coli and yeast
Contains a gene URA3 (uracil production pathway enzyme)

37. Ti Plasmid From soil microorganism Abgrobacterium Tumefaciens.
For plant transformation
Ti plasmid contains T-DNA which integrates into host genome
Binary vector are now developed on it
Integrates into host genome for stable transformation