DNA Cloning Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations.

Cloning of any DNA fragment essentially involves four steps 1. fragmentation - breaking apart a strand of

DNA 2. ligation - gluing together pieces of DNA in a

desired sequence 3. transfection - inserting the newly formed

pieces of DNA into cellsscreening/selection - selecting out the cells that were successfully transfected with the new DNA

Molecular biologists adopted the pure enzymes as tools for manipulating DNA molecules in pre-determined ways, using them to make copies of DNA molecules, to cut DNA molecules into shorter fragments, and to join them together again in combination that do not exist in nature

Recombinant DNA methodology led to the development of DNA or gene cloning, in which short DNA fragments, possibly containing a single gene, are inserted into a plasmid or virus chromosome and then replicated in a bacterial or eukaryotic host

Enzymes for DNA manipulation DNA polymerase which are enzymes that synthesize new polynucleotides complementary to a existing DNA or RNA template

Nuclease which degrade DNA molecules by breaking the phophodiester bonds that link one nucleotide to the next

Ligases which join DNA molecules together by synthesizing phosphodiester bonds between nucleotides at the ends of a single molecule

End-modification enzymes which make changes to the ends of DNA molecules, adding an important dimension to the design of ligation experiments, and providing one means of labeling DNA molecules with radioactive and other markers.

DNA polymerase

A DNA polymerase requires a primer in order to initiate the synthesis of a new polynucleotide. The sequence of this oligonucleotide determines the position at which it attached to the template DNA and hence specifies the region of the template that will be copies. When a DNA polymerase is used to make new DNA in vitro, the primer is usually a short oligonucleotide made by chemical synthesis

Nuclease

The cut are made in slightly different position relative to the recognition sequence

Restriction endonucleases enable DNA molecules to be cut at defined positions

Restriction enzymes cut DNA in two different ways. 1) Blunt end 2) Sticky or cohesive end

DNA ligase

DNA fragments that have been generated by treatment with a restriction endonuclease can be joined back together again, or attached to a new partner, by a DNA ligase. The reaction requires energy, which is provided by adding either ATP or NAD to the reaction mixture.

Sticky-end ligation

Blunt End Ligation

The greater efficiency of sticky-end ligation has stimulated the development of methods for converting blunt ends to sticky ends. In one method, short double-stranded molecules called linkers or adaptors are attached to the blunt ends. Linkers and adaptors work in slightly different ways but both contain a recognition sequence for a restriction endonuclease and so produce a sticky end after treatment with the appropriate enzyme.

DNA cloning If the recombinant plasmid is reintroduced into E. coli, and the inserted gene has not disrupted its replicative activity, then the plasmid plus inserted gene will be replicated and copies passed to the daughter bacteria after cell division. More rounds of plasmid replication and cell division will result in a colony of recombinant E.coli. Bacteria, each bacterium containing multiple copies of the animal gene. This series of events, as illustrated in this figure, constitutes the process called DNA or gene cloning

Plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.

Cloning Vector

The plasmid acts as a cloning vector, providing the replicative ability that enables the cloned gene to be propagated inside the host cell. Plasmids replicate efficiently in bacterial hosts because each plasmid possesses an origin of replication which is recognized by the DNA polymerases and other proteins that normally replicate the bacterium’s chromosome

This vector carries the ampicillin-resistance gene from pBR322, along with a second gene, called lacZ’, which is part of the E.coli gene for the enzyme β-galactosidase. The remainder of the lacZ gene is located in the chromosome of the special E. coli strain that is used when cloning genes with pUC8. The proteins specified by the gene segments on the plasmid and on the chromosome are able to combine to produce a functional β-galactosidas enzyme. The presence of functional β-galactosidas molecules in the cells can be checked by a hostochemical test with a compound called X-gal (5-bromo-4-chloro-3-indoly-β-D-galactopyranoside), which the enzyme converts into a blue product. All colonies that grow on this medium are made up of transformed cells because only transformants are ampicillin resistant. Some colonies are blue and some are white. The white colonies disrupted lacZ’ genes; these are recombinats

Cloning vectors based on E.coli bacteriophage

The first attempts to develop vectors able to handle larger fragments of DNA centered on bacteriophage λ. The λ genome is able to integrate into the bacterial chromosome, where it can remain quiescent for many generations, being replicated along with the host chromosome every time the cell divides. This is called the lysogenic infection cycle

Treatment with the appropriate restriction endonuclease produces the left and right arms, both of which have one blunt end and one end with the 12-nucleotide overhang of the cos site. The DNA to be cloned is blunt ended and so is inserted between the two arms during the ligation step. These arms also ligate to one another via their cos sites, forming concatamer. Some parts of the concatamer comprise left arm-insert DNA-right arm and, assuming this combination is 37-52 kb in length, will be enclosed inside the capsid by the in vitro packaging mix. Parts of the concatamer made up of left arm ligated directly to right arm, without new DNA, are too short to be packaged.

Lambda Phage Used for Making Genome Libraries

Cosmid Libraries Used for larger Genomic Sequences : hybrid derived from plasmid and λ phages

YAC: Yeast Artificial Chromosome

Natural yeast chromosomes range from 230 kb to over 1700 kb, so YACs have the potential to clone Mb-sized DNA fragments. This potential has been realized, standard YACs being able to clone 600 kb fragments, with special types able to handle DNA up to 1400 kb in length. Currently this is the highest capacity of any type of cloning vector, and several genome projects have made extensive use of YAC

Important Basic Techniques in Analysis of RNA & DNA

1) Agarose and Polyacrylamide Gel Electrophoresis of Nucleic Acids.

2) Molecular Hybridization to Determine RNA and DNA Relatedness.

3) Development of Plasmids and Viruses As Cloning Vehicles.

4) Use of Restriction Enzymes for DNA Analyses

5) Polymerase Chain Reaction for Amplifying Nucleic Acids.

6) Techniques for Sequencing DNA and RNA.

7) Mutagenesis to Make Precise Changes to Nucleic Acids.

Northern (RNA) Blot Hybridization Used for Detection of mRNAs.

Eth Brom T N A

1) Separate RNA on an Agarose Gel. 2) Stain Gel With Ethidium Bromide 3) Photograph RNA Under UV Light. 4) Wick to Nitrocellulose in Salt Buffer. 5) Hybridize RNA With 32P- labelled Probes. 6) Wash & Expose to X-ray Film.

Northern Hybridization is useful for identifying specific RNAs & determining their abundance.

Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae

Gene Chips Can Be Used for Detection of Hundreds of mRNAs.

DNA chip showing colored spots specific for hybridization with different mRNAs in experimental and control tissue. The intensity of the spots reflects abundance of the RNAs and the color shows the relative ratios of the RNAs.

1) Microarray chips contain 10,000 - 100,000 DNA oligonucleotide spots representing the mRNAs in a cell.

2) RNAs from different treatments are purified and used as probe sources.

3) Reverse transcriptase is used to label cDNAs differentially with colored fluorescent nucleotides.

4) The fluorescent probes are mixed & hybridized to the gene chip DNAs. Hybridizations are proportional to the relative abundance of the RNAs.

5) The intensity and colors of the spots is recorded by a microarray machine that provides a printout of the data and numerical analysis of the fluorescence.

6) This data is used to calculate the relative amounts of each mRNAs accumulating with different experiments.

PCR - The Polymerase Chain Reaction 1) Heat DNA to denature base pairing to

produce single strands. 2) Cool slowly to anneal the synthetic primers

to defined the region to be amplified. 3) Copy DNA strands with a heat stable DNA

polymerase to form two double strands. 4) The copied strands can be amplified many

times by repeating steps 1 to 3.

PCR Is Extremely Useful for DNA Cloning, Mapping and Sequencing.

Can Start With Extremely Small Amounts of Impure Material & Amplify Very Large Amounts of Pure DNA After 30 - 40 Cycles.

Southern (DNA) Blot Hybridization

1) Isolate DNA of Interest & Digest with Restriction Enzymes. 2) Separate Individual DNA Fragments On An Agarose Gel. 3) Denature the Separated DNA with Alkali (0.5 M NaOH). 4) Blot or Transfer the DNA to Nitrocellulose or Nylon Filters. 5) Hybridize with Radioactive Nucleic Acid Probes.

6) Identify Hybridized DNA Species by Autoradiography.

Southern Blots Provide a Powerful Tool for DNA Mapping

DNA Fingerprint Detection

Restriction Endonucleases

1) Recognize double stranded DNA at specific nucleotide target sites.

2) Target sites are palindromes. 3) Cleave DNA leaving 3’or 5’ sticky

overhanging ends or a blunt end sequence.

4) Literally hundreds of individual enzymes with different target specificities have been isolated from a wide range of microbes.

5) Form the basis for recombinant DNA technology because the ends can be rejoined by nonspecific DNA ligases.

6) Restriction Enzymes form the basis for precise mapping of DNA from related organisms.

Plasmid DNA Fragments produced by different Enzymes

Discovery of Restriction Enzymes is One of the Most Important Biological Findings of the Century.

Introduction to Genomics

Important steps preceding development of value-added products

What we need……..?

A well characterized gene/genes with known function

How ……..?

Power of Genomics….

What is genomics?

The study of the organization, expression, regulation, interaction and function of the entire genetic complement of an organism

Genomics is the discovery and study of many genes simultaneously on genome –wide scale

Genomics Characterization of the entire genetic

Organization of an organism

Functional Genomics Characterization of the overall profile

of gene expression

Proteomics Characterization of the array of protein

Function and interaction

Primary approaches of genomics

Metabolomics The total metabolite composition (the metabolome) of an organism.

Genomics Can be….

Structural Genomics (Where It is?) Sequence ESTexpressed sequence tag s

Genome sequence Genome organization Functional genomics (What it does?) Gene index Microarray Expression profiling DNA chip Silencing Comparative genomics Bioinformatics

Genomics processes involved in gene identification

• Mapping - genetic linkage mapping - physical mapping Map based cloning of genes

• Sequencing

• Determination of function - bioinformatics - functional genomics

- Knockouts - microarrays - Proteomics

• Past – Genetic maps

Distance between simple markers expressed in units of recombination

– Cytological maps Stained chromosomes, observable under microscope

• Present – Physical maps

Distance between nucleotides expressed in bases

– Comparative map Corresponding genes detection; Regulatory sequence detection;

Mapping Genomes

Genomics processes involved in gene identification

Physical Mapping

Determination of the actual physical distance between loci

Clone contig In situ hybridization

Genomics Activities

• Many complete genomes have now been sequenced

• TIGR presents a list of publicly available genomic projects (This list counts individual eukaryotic chromosomes separately.)

As of September 2004, there were 163 complete genomes publicly available via TIGR

• Many more genomes have been sequenced, but are held in the private sector

• Genomes vary tremendously in size, and not in a way that is easily predictable

All of the genome sizes you ever wanted to know

Smallest plant genome

Smallest cereal genome

Why study complete Genomes?

• Determine what is missing from the genome • Identify genes

• Study non-coding regions of the genome Introns, promoters, telomeres, etc.

We probably are not yet aware of all regulatory and structural features found in genomes

• Provide large databases that are amenable to statistical methods • Identify variant sequences that may have subtle phenotypes • Study evolution of the organism and genome

Genome Sequencing

Whole genome is fragmented in moderate sized pieces and cloned Genetic and physical markers are used to order the clones Sequences are assembled into genome based on overlap

Bioinformatics

Computational or algorithmic approaches to the production of information from large amounts of biological data, include prediction of protein

structure, dynamic modeling of complex physiological systems or the statistical treatment of quantitative

traits in populations in order to determine the genetic basis for these traits.

Bioinformatics is the acquisition, curation and interrogation of large collections of complex biological data

Sequence Identification

BLAST: Basic Local Alignment Search Tool Program for identifying database Sequence with similarity to a Query sequence Most frequently access through NCBI

Sequence Identification

E value: probability that the given match occurs by chance 2e-31: the probability here in this case is 0 .000000000000000000000000000000 Due to random match

Comparative Genomics

The genomes of several grasses arranged so that regions carrying similar genes are aligned

Gale and Devos 1998, PNAS, vol 95 :1971-1974

Need for Comparative Studies

• Identify core set of genes for all organisms • Identify contents of ancestral genome • Is there any real 'model organism'? If so, what is it? • Do all organisms use the same gene for the same purpose?

Single Nucleotide Polymorphism

Inter-genic regions Coding regions Every 1400bp Every 1430bp

Single nucleotide polymorphisms (SNPs) account for most of the genetic differences between individuals

SNPs in human population

Example: Sickle Cell Anemia

SNP on Beta Globin gene, which is recessive: Sickle looks like this:

Functional Genomics

Determination of gene function

• Gene inactivation- “ knockouts” - Insertional mutagenesis

- T-DNA or transposons - RNAi

• Gene overexpression - RT-PCR - microarrays • Proteomics

Determining the Function of Genes Through Gene Disruption (Knockouts)

• Barbara McClintock was the first scientist to predict that transposable elements (mobile DNA)

are present in eukaryotic genomes

Transposable Elements

A Two Component Ac- Ds System

Ds Ds pSP-Ds-Ubi-bar (5.95 kb)

Ubi

1

bar

nos

5’ 3’

pSP

pSP

5’

Cod

A

Act1

Ubi

1

35S

AcTPase

nos

pBS-codA-Act-UbiAc (11.6 kb) Footprints

•Transposase

•Jump

•Landing

Development of Ds transposon insertion lines in barley

Line 1 Line 2

Barley line with active transposase

Cod

A

x Barley line containing

transposed Ds

Ds transposition

Stable single copy Ds transposants

Selection for non AcTPase, Ds-containing plants

3’

Ubi

1

bar

nos

5’

(32B-1) Ac

t1

Ubi

1

35S

AcTPase

nos

5’ 3’ Ds Ds

DNA was digested with EcoRV and probed with Ds 5’ element (400 bp)

A scheme for generating secondary Ds insertion lines

~ 100 TNP lines have been generated

Ds

Ubi

Ds

bar nos Nco1 Nco1 Nco1

Ds

Ubi Nco1 Nco1

Self Ligation

Digestion with Nco1

Ds

Nco1

Ubi

P1 P2 P3 P4

Ds bar r

nos

Nco1

Ubi

P5 P6 P8 P7

Ds

bar nos

Nco1 Nco1

Nested-iPCR

5’ 3’

3’ 5’

5’ 3’

PCR tools to identify genes disrupted by transposon

Expressed Sequence Tags (ESTs)

Wall-associated protein kinase (722 aa)

70 100 650

ATP-binding

900

Active-site

1.5 kb 1.2 kb Ds

1263

Calcium-binding EGF-like domain

700 bp 600 bp

P450 cytochrome (543 aa) P450 domain

Ds

187 389 150 248

Ds Elements Move Into Genes

Exon Intron Domain Ds

Over-expression of genes

Control Over-expressor

0%

50%

100%

150%

200%

250%

Fresh Weight

Dry Weight

Seed Yield

% I

ncre

ase

Control Over-expressor line 1 Over-expressor line 2

Mendel has identified 18 transcription factor genes that

regulate plant growth. Several studies have identified many transcription

factor genes that regulate plant growth.

116.3

12%

S D S P A G E

4 7 pH 4 7 pH 200

97.4 66.3 55.4

36.5

31

21.5

14.4

TNP 4-Ds mutant GP-wild type

Protein Expression

Knock out using Plant Virus

Transcriptome

Proteome

Gene expression

Genome DNA

RNA

Proteins Gene translation

Diverse metabolic pathways

Phenome

Functional Genomics

Metabolome Metabolites

Integration of Genomics Tools can Unravel Genome and Complex Biological Relationships