An introduction to recombinant DNA technology

S.K.Kashyap

contents

1. rDNA technology makes it possible to clone genes for basic research and commercial applications: an overview 2. Restriction enzymes are used to make recombinant DNA 3. Genes can be cloned in recombinant DNA vectors: a closer look 4. Cloned genes are stored in DNA libraries 5. The polymerase chain reaction (PCR) cloned DNA directly in vitro

The mapping and sequencing of the human genome has been made possible by advances in DNA technology.

Progress began with the development of techniques for making recombinant DNA, in which genes from two different sources - often different species - are combined in vitro into the same molecule.
These methods form part of genetic engineering, the direct manipulation of genes for practical purposes.

Applications include the introduction of a desired gene into the DNA of a host that will produce the desired protein.

DNA technology has launched a revolution in biotechnology, the manipulation of organisms or their components to make useful products.

Practices that go back centuries, such as the use of microbes to make wine and cheese and the selective breeding of livestock, are examples of biotechnology. Biotechnology based on the manipulation of DNA in vitro differs from earlier practices by enabling scientists to modify specific genes and move them between organisms as distinct as bacteria, plants, and animals.

DNA technology is now applied in areas ranging from agriculture to criminal law, but its most important achievements are in basic research.

Cloning - a definition
From the Greek - klon, a twig An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody) An individual grown from a single somatic cell of its parent & genetically identical to it Clone: a collection of molecules or cells, all identical to an original molecule or cell

DNA CLONING

A method for identifying and purifying a

particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest.

Gene cloning
When DNA is extracted from an organism, all its genes are obtained In gene (DNA) cloning a particular gene is copied (cloned)

Why Clone DNA?

A particular gene can be isolated and its nucleotide sequence determined Control sequences of DNA can be identified & analyzed Protein/enzyme/RNA function can be investigated Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be engineered for specific purposes, e.g. insulin production, insect resistance, etc.

Sources of DNA for Cloning

1) Chromosomal DNA 2) RNA converted to cDNA 3) chemically synthesized genes 4) PCR-amplified DNA

Molecular Cloning

In order to have enough DNA to work with for a single gene or sequence, you must have a way to clone, or reproduce many exact copies of that gene. This is called molecular cloning The DNA containing the gene must be isolated away from its genomic context That should be simple but there are big problems

Gene of interest

To study a particular gene, scientists needed to develop methods to isolate only the small, well-defined, portion of a chromosome containing the gene.

Techniques for gene cloning enable scientists to prepare multiple identical copies of gene-sized pieces of DNA.

An overview

The potential uses of cloned genes fall into two general categories. First, the goal may be to produce a protein product.

For example, bacteria carrying the gene for human growth hormone can produce large quantities of the hormone for treating stunted growth.

Alternatively, the goal may be to prepare many copies of the gene itself.

This may enable scientists to determine the genes nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.

Gene cloning and genetic engineering were made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations. In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria. Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.

Bacteria protect their own DNA by methylation.

Restriction endonucleases

Also called restriction enzymes Occur naturally in bacteria Hundreds are purified and available commercially Named for bacterial genus, species, strain, and type Example: EcoRI
Genus: Escherichia Species: coli Strain: R

Restriction endonucleases

Recognize specific base sequences in DNA Cut DNA at those recognition sites

Restriction Enzyme Recognition Site

Enzymes recognize specific 4-8 bp sequences
EcoRI 5GAATTC3 3CTTAAG5

Recognition sites have symmetry Some enzymes cut in a staggered fashion Some enzymes cut in a direct fashion
PvuII 5CAGCTG3 3GTCGAC5

Products generated by restriction enzymes

COHESIVE END CUTTERS (staggered cuts): Enzyme Recognition Site Ends of DNA After Cut EcoRI 5GAATTC3 3CTTAAG5 5CTGCAG3 3GACGTC5 5G 3CTTAA 5CTGCA 3G AATTC3 G5 G3 ACGTC5

PstI

BLUNT END CUTTERS (direct cuts): Enzyme Recognition Site Ends of DNA After Cut HaeIII 5GGCC3 3CCGG5 5GG 3CC CC3 GG5

Frequency of cutting

Average distance between cuts is:

4n
where n is number of bps in recognition site.

4-base cutter: 5-base cutter: 6-base cutter: 8-base cutter:

44 = 256 bp 45 = 1,024 bp 46 = 4,096 bp 48 = 65,536 bp

Each restriction enzyme cleaves a specific sequences of bases or restriction site.

These are often a symmetrical series of four to eight bases on both strands running in opposite directions.

If the restriction site on one strand is 5, the complementary strand is 3.

3-CTTAGG-

5-GAATTC-

Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.

Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme.

Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a staggered way creating single-stranded ends sticky ends.

These extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme.

These DNA fusions can be made permanent by DNA ligase which seals the strand by catalyzing the formation of phosphodiester bonds.

Restriction enzymes and DNA ligase can be used to make recombinant DNA, DNA that has been spliced together from two different sources.

3. Genes can be cloned in DNA vectors: a closer look

Recombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids.

These can be returned relatively easily to bacteria. The original plasmid used to produce recombinant DNA is called a cloning vector, which is a DNA molecule that can carry foreign DNA into a cell and replicate there.

Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it.

 Bacteria are most commonly used as host cells for gene cloning because DNA can be easily isolated and reintroduced into their cells. Bacterial cultures also grow quickly, rapidly replicating the foreign genes.

The process of cloning a human gene in a bacterial plasmid can be divided into five steps.

1. Isolation of a vector and gene-source DNA.

The source DNA comes from human tissue cells. The source of the plasmid is typically E. coli.

This plasmid carries two useful genes:

ampR, conferring resistance to the antibiotic ampicillin lacZ, encoding the enzyme beta-galactosidase which catalyzes the hydrolysis of sugar.

The plasmid has a single recognition sequence, within the lacZ gene, for the restriction enzyme used.

2. Insertion of DNA into the vector.

By digesting both the plasmid and human DNA with the same restriction enzyme we can create thousands of human DNA fragments, one fragment with the gene that we want, and with compatible sticky ends on bacterial plasmids. After mixing, the human fragments and cut plasmids form complementary pairs that are then joined by DNA ligase. This creates a mixture of recombinant DNA molecules.

3. Introduction of the cloning vector into cells.

Bacterial cells take up the recombinant plasmids by transformation.

These bacteria are lacZ-, unable to hydrolyze lactose.

This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA, both recombinant and nonrecombinant.

STEP 4. TRANSFORMATION OF LIGATION PRODUCTS

The process of transferring exogenous DNA into cells is call transformation There are basically two general methods for transforming bacteria. The first is a chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells. A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake.

CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

TRANSFORMATION BY ELECTROPORATION

STEP 5. GROWTH ON AGAR PLATES

4. Cloning of cells (and foreign genes).

We can plate out the transformed bacteria on solid nutrient medium containing ampicillin and a sugar called X-gal.

Only bacteria that have the ampicillin-resistance plasmid will grow. The X-gal in the medium is used to identify plasmids that carry foreign DNA.

Bacteria with plasmids lacking foreign DNA stain blue when beta-galactosidase hydrolyzes X-gal. Bacteria with plasmids containing foreign DNA are white because they lack beta-galactosidase.

5. Identifying cell clones with the right gene.

In the final step, we will sort through the thousands of bacterial colonies with foreign DNA to find those containing our gene of interest. One technique, nucleic acid hybridization, depends on base pairing between our gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule.

The sequence of our RNA or DNA probe depends on knowledge of at least part of the sequence of our gene. A radioactive or fluorescent tag labels the probe.

The probe will hydrogenbond specifically to complementary single strands of the desired gene.

After denaturation (separating) the DNA strands in the plasmid, the probe will bind with its complementary sequence, tagging colonies with the targeted gene.

Because of different details between prokaryotes and eukaryotes, inducing a cloned eukaryotic gene to function in a prokaryotic host can be difficult.

One way around this is to employ an expression vector, a cloning vector containing the requisite prokaryotic promotor upstream of the restriction site. The bacterial host will then recognize the promotor and proceed to express the foreign gene that has been linked to it, including many eukaryotic proteins.

The presence of introns (the non-coding regions) in eukaryotic genes creates problems for expressing these genes in bacteria.

To express eukaryotic genes in bacteria, a fully processed mRNA acts as the template for the synthesis of a complementary strand using reverse transcriptase. This complementary DNA (cDNA), with a promoter, can be attached to a vector for replication, transcription, and translation inside bacteria.

 Complementary DNA is DNA made in vitro using mRNA as a template and the enzyme reverse transcriptase.

Molecular biologists can avoid incompatibility problems by using eukaryotic cells as host for cloning and expressing eukaryotic genes. Yeast cells, single-celled fungi, are as easy to grow as bacteria and have plasmids, rare for eukaryotes. Scientists have constructed yeast artificial chromosomes (YACs) - an origin site for replication, a centromere, and two telomeres with foreign DNA. These chromosomes behave normally in mitosis and can carry more DNA than a plasmid.

Another advantage of eukaryotic hosts is that they are capable of providing the posttranslational modifications that many proteins require.

This includes adding carbohydrates or lipids. For some mammalian proteins, the host must be an animal or plant cell to perform the necessary modifications.

Many eukaryotic cells can take up DNA from their surroundings, but often not efficiently.
Several techniques facilitate entry of foreign DNA.

In electroporation, brief electrical pulses create a temporary hole in the plasma membrane through which DNA can enter. Alternatively, scientists can inject DNA into individual cells using microscopically thin needles. In a technique used primarily for plants, DNA is attached to microscopic metal particles and fired into cells with a gun. Once inside the cell, the DNA is incorporated into the cells DNA by natural genetic recombination.

4. Cloned genes are stored in DNA libraries

In the shotgun cloning approach, a mixture of fragments from the entire genome is included in thousands of different recombinant plasmids. A complete set of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome, forms a genomic library.

The library can be saved and used as a source of other genes or for gene mapping.

A more limited kind of gene library can be developed from complementary DNA (cDNA).

During the process of producing cDNA, all mRNAs are converted to cDNA strands.

This cDNA library represents that part of a cells genome that was transcribed in the starting cells.
This is an advantage if a researcher wants to study the genes responsible for specialized functions of a particular kind of cell. By making cDNA libraries from cells of the same type at different times in the life of an organism, one can trace changes in the patterns of gene expression.

cDNA synthesis
TTTTTTTTT

First strand

Nick translation

cDNA library

EcoR I

Infect cells

Genomic Library

AAAAAA AAAAAA AAAAAA AAAAAA

AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA

AAAAAA AAAAAA AAAAAA AAAAAA

AAAAAA AAAAAA

AAAAAA AAAAAA
AAAAAA

cDNA synthesis

cDNA library

Infect cells

Genomic library

cDNA library
mRNA Species or strains Tissues Developmental stages 0.2k -- 6k Correlate with expression level Expression vs. non expression DNA or antibody or protein Encoded protein Infer protein identity

Source
Variation

Genomic DNA Species or strains

Insert size Representation Type

12k -- 20k Equal

Only one DNA Gene structure Infer protein identity

Probe
Purpose

Subcloning of the cDNA insert

EcoR I EcoR I EcoR I

Is there a better way to subclone the insert?

In addition to plasmids, certain bacteriophages are also common cloning vectors for making libraries.

Fragments of foreign DNA can be spliced into a phage genome using a restriction enzyme and DNA ligase. The recombinant phage DNA is packaged in a capsid in vitro and allowed to infect a bacterial cell. Infected bacteria produce new phage particles, each with the foreign DNA.

Whats the trouble with isolating a gene?

All genes are made of the same molecule that is chemically quite homogeneous (DNA all As,Cs, Gs and Ts) There is a huge amount of genetic material E. coli >1000 genes (4,600,000 bp) Humans perhaps 30,000 genes (app. 4,400,000,000 bp)

Single genes only make a tiny fraction of the whole genome

In E. coli, each gene is app. 0.05 % of the genome In humans, each gene is app. .00005% of the genome Finding a gene is not like like looking for a needle in a haystack, it is more like looking for one particular piece of hay in a haystack! In this case all the hay looks the same, and you can only tell the difference by analyzing the chemistry of each piece!

Break the genome into more manageable pieces

Restriction enzymes - isolated from bacteria probably involved in protecting the cell from viral foreign DNA Example: EcoRI from E. coli Cleaves six base pair site E. coli DNA is protected by a corresponding DNA methylase that adds methyl groups to the central base pairs and prevents cleavage

These enzyme are purified and used as tools in vitro Will cleave DNA at any GAATTC sequence - every genome has a specific pattern of GAATTC sequences that occurs at random, but is always the same

How do you stably maintain and replicate a foreign DNA fragment? by hitching a ride on a stable replicon
Properties of plasmid cloning vector 1. Small 2. Stable in the chosen host usually E. coli 3. High copy number 4. Easy to purifiy 5. Can accommodate foriegn DNA 6. Single cloning sites 7. Selectable marker antibiotic resistance 8. Easily introduced into host (transformation or transduction

Simple fusion of different DNA fragments

EcoRI EcoRI

pBR322, cut at its single Eco RI site

DNA ligase EcoRI foreign DNA EcoRI recombinant DNA New Eco RI site CNNN TTAAGNNN EcoRI end on vector NNNGAATTCNNN NNNCTTAAGNNN Fusion site

Sticky ends NNNGAATT NNNC EcoRI end on insert

a-complementation relies on modular structure of bgalactosidase

- basic idea is often used with cloning vectors called insertional inactivation

a-complementation
LacZ+ - blue colony LacZ- - while colony -if you interrupt the lacZ gene, the colony is white

How does acomplementation work?

It all comes down to bgalactosidase
Certain strains supply the b-Gal fragment When a is supplied in trans, this allows b-Gal to function

One major issue is the size of the insert DNA

The larger the fragment, the more of the source genome can be represented on one plasmid For a gene library, we can easily calculate the number of individual recombinant molecules required to represent an entire genome

N = [ln (1-P)]/[ln (1-f)]

P probability of complete coverage N number of individual clones required f proportion of genome in average fragment

For the human genome and a standard plasmid P 99% or 0.99 confidence f 5 kb/4,400,000 kb = 1.14 x 10-6

N = [ln .01]/[ln 0.99999886] = -4.6/-1.14 x 10-7 = 40,350,877 individuals Plasmid vectors stable inserts of 5 kb

Bacteriophage vectors can acommodate larger inserts

larger inserts than plasmids introduced by phage infection of cells 20 35 kb inserts

For the human genome and a standard plasmid P 99% or 0.99 confidence f 35 kb/4,400,000 kb = 7.95 x 106

N = [ln .01]/[ln 0.99999205] = -4.6/-7.95 x 10-6 = 578, 616 individuals

A big improvement!

Yeast artificial chromsomes (YACs) and bacterial artificial chromsomes (BACs)

Can accomodate from 300 500 kb of DNA great for large genomes

For the human genome and a standard plasmid P 99% or 0.99 confidence f 500 kb/4,400,000 kb = 1.14 x 10-4

N = [ln .01]/[ln 0.999886] = -4.6/-1.14 x 10-4 = 40,350 individuals

A even bigger improvement!

Whats a YAC? Yeast artificial chromsome

self-replicating vector that can be maintained in yeast Can accommodate large insert fragments

Reeves et al., 1992, Methods Enzymol. 216:584-603

What are BACs? bacterial artificial chromsomes

- very low copy number vectors that can accomodate huge inserts

Derived from the F plasmid of E. coli - very stably maintained - 1-2 copies per cell (strict copy number contro)

Shizuya et al, 1992, PNAS 89:9794-8797

A wide range of BAC libraries are now available for the human genome as well as other large genomes

Human Source BAC material libraries A 987SK cells 987SK cells

Plate numbers 1-1,000 (96well) 1-194 (384well)

human 195-879 (384sperm well) (HSP) human sperm (HSP) 2,001-2,423 (384-well)

D1

human sperm (HSP) human sperm (HSP) human sperm (HSP)

2,501-2,565 (384-well)

D2

2,566-2,671 (384-well) 3,000-3,253 (384-well)

Caltech & Average NHGRIAvailable Cloning Constructed GDB Insert DOE from Site by Names Size approval Tatiana CIT978SKSlepak, 1A1 to 115 kb Caltech HindIII Valeria 1000H12 Mancino CIT978SKResearch Yu-Ling 1001A1 to 120 kb HindIII Genetics Sheng 1194P24 CIT-HSPResearch Yu-Ling 1195A1 to 125 kb HindIII Genetics Sheng 1900P24 Caltech, Genome Yu-Ling 129 kb Systems, HindIII Approved Sheng, YuResearch Jiun Chen Genetics Caltech, Genome 202 kb Systems, EcoRI Approved Research CIT-HSPGenetics 2000A1 and up Research 182 kb EcoRI Approved Genetics Sangdun Choi Research 142 kb EcoRI Approved Genetics

3,254human transforamtion sperm on-going (HSP) (384-well)

166 kb

EcoRI Approved