Download ppt - UNIT 5 Recombinant DNA Technology. Objectives Define Recombinant DNA technology Define Recombinant DNA technology Outline steps involved in creating recombinant

UNIT 5UNIT 5

Recombinant Recombinant DNA TechnologyDNA Technology

ObjectivesObjectives Define Recombinant DNA technologyDefine Recombinant DNA technology Outline steps involved in creating recombinant DNA Outline steps involved in creating recombinant DNA Define type II restriction enzymes and their use in Define type II restriction enzymes and their use in

recombinant DNA technologyrecombinant DNA technology Explain restriction mapping and construct restriction Explain restriction mapping and construct restriction

mapsmaps State the properties of cloning vectors and reproduce State the properties of cloning vectors and reproduce

genetic map of common cloning vectorsgenetic map of common cloning vectors Outline the steps involved in cloning prokaryotic DNA Outline the steps involved in cloning prokaryotic DNA

into a plasmid cloning into a plasmid cloning vectorvector Outline the steps involved in cloning eukaryotic DNA Outline the steps involved in cloning eukaryotic DNA

into a plasmid cloning into a plasmid cloning vectorvector Understand the methods of transformation and the Understand the methods of transformation and the

steps involved in the selection of transformantssteps involved in the selection of transformants Explain the concept of gene libraries and methods of Explain the concept of gene libraries and methods of

screening a gene libraryscreening a gene library

Recombinant DNA Recombinant DNA technologytechnology

a body of techniques for cutting and a body of techniques for cutting and splicing together different pieces of DNA. splicing together different pieces of DNA.

When segments of foreign DNA are When segments of foreign DNA are transferred into another cell or organism, transferred into another cell or organism, the protein for which they code may be the protein for which they code may be produced along with substances coded produced along with substances coded for by the native genetic material of the for by the native genetic material of the cell or organism. cell or organism.

These cells become "factories" for the These cells become "factories" for the production of the protein coded for by the production of the protein coded for by the inserted DNAinserted DNA

Restriction EnzymesRestriction Enzymes

The major tools of recombinant DNA The major tools of recombinant DNA technology are technology are restriction enzymes restriction enzymes (nucleases)(nucleases) first discovered in the late 1960sfirst discovered in the late 1960s They work by cutting up the foreign DNA, a They work by cutting up the foreign DNA, a

process called restriction. process called restriction. Most restriction enzymes are very specificMost restriction enzymes are very specific recognizing short, specific nucleotide recognizing short, specific nucleotide

sequences in DNA molecules and cutting at sequences in DNA molecules and cutting at specific points within these sequences. specific points within these sequences.

There are hundreds of restriction enzymes and There are hundreds of restriction enzymes and more than 150 different recognition more than 150 different recognition sequences.sequences.

Restriction enzymesRestriction enzymes

Nucleases are further described by Nucleases are further described by addition of the prefix "endo" or "exo" to addition of the prefix "endo" or "exo" to the name: the name:

endonuclease applies to sequence specific endonuclease applies to sequence specific nucleases that break nucleic acid chains nucleases that break nucleic acid chains somewhere in the interior, rather than at the somewhere in the interior, rather than at the ends, of the molecule.ends, of the molecule.

exonucleases function by removing exonucleases function by removing nucleotides from the ends of the molecule nucleotides from the ends of the molecule

Types of Restriction Types of Restriction EnzymesEnzymes

Type IType I Recognise specific sequences·but then track Recognise specific sequences·but then track along DNA (~1000-5000 bases) before cutting one of along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the endonuclease is required to cut the 2nd strand of the DNA the DNA

Type IIType II Recognise a specific target sequence in Recognise a specific target sequence in DNA, and then break the DNA (both strands), within DNA, and then break the DNA (both strands), within the recognition site the recognition site

Type III Type III Intermediate properties between type I Intermediate properties between type I and type II. Break both DNA strands at a defined and type II. Break both DNA strands at a defined distance from a recognition site distance from a recognition site

Restriction Restriction EndonucleasesEndonucleases

The first endonucleases discovered was from The first endonucleases discovered was from Escherichia coli Escherichia coli EcoRIEcoRI

A restriction endonuclease functions by A restriction endonuclease functions by "scanning" the length of a DNA molecule. "scanning" the length of a DNA molecule.

Once it encounters its particular specific Once it encounters its particular specific sequence (sequence (recognition siterecognition site), it will bond to the ), it will bond to the DNA molecule and makes one cut in each of the DNA molecule and makes one cut in each of the two sugar-phosphate backbones of the double two sugar-phosphate backbones of the double helix.helix.

Once the cuts have been made, the DNA Once the cuts have been made, the DNA molecule will break into fragments. molecule will break into fragments.


The positions of these two cuts, both in relation to The positions of these two cuts, both in relation to each other, and to the recognition sequence itself, each other, and to the recognition sequence itself, are determined by the identity of the restriction are determined by the identity of the restriction endonuclease endonuclease

The length of the recognition site for different The length of the recognition site for different enzymes can be four, five, six, eight or more enzymes can be four, five, six, eight or more nucleotide pairs long. nucleotide pairs long.

Restriction endonucleases that cleave recognition Restriction endonucleases that cleave recognition sites of four and six nucleotide pairs are used for sites of four and six nucleotide pairs are used for most of the protocols for molecular cloning since most of the protocols for molecular cloning since these restriction sites are common in DNA.these restriction sites are common in DNA.


Different endonucleases yield different sets Different endonucleases yield different sets of cuts, but one endonuclease will always cut of cuts, but one endonuclease will always cut a particular base sequence the same way.a particular base sequence the same way.

Some restriction endonucleases digest DNA Some restriction endonucleases digest DNA leaving 5′-phosphate extensions(protruding ends, leaving 5′-phosphate extensions(protruding ends, sticky ends)sticky ends)

Some leave 3′-hydroxyl extensionsSome leave 3′-hydroxyl extensions

Some cut the backbone of both strands within a Some cut the backbone of both strands within a recognition site to produce blunt-ended (flush-recognition site to produce blunt-ended (flush-ended) DNA molecules. ended) DNA molecules.

Use of Restriction Enzymes Use of Restriction Enzymes in recombinant DNA in recombinant DNA

TechnologyTechnology

How Restriction Enzymes How Restriction Enzymes are namedare named

generally have names that reflect their generally have names that reflect their origin—origin— The first letter of the name (capitalized ) The first letter of the name (capitalized )

comes from the genus of the bacteria comes from the genus of the bacteria the second two letters (lower case) come the second two letters (lower case) come

from the species from the species Roman numerals following the nuclease Roman numerals following the nuclease

names indicate the order in which the names indicate the order in which the enzymes were isolated from that strain of enzymes were isolated from that strain of bacteria.bacteria.

For example For example EcoRIEcoRI comes from comes from Escherichia Escherichia colicoli RY13 RY13

Type II Restriction Type II Restriction EnzymesEnzymes

Many Type II restriction endonucleases recognise Many Type II restriction endonucleases recognise PALINDROMIC PALINDROMIC sequencessequences Symmetrical sequences which read in the same order of Symmetrical sequences which read in the same order of

nucleotide bases on each strand of DNA (always read 5' nucleotide bases on each strand of DNA (always read 5' to 3') to 3')

For example, For example, EcoEcoRI recognises the sequence RI recognises the sequence 5'-G A A T T C-3'5'-G A A T T C-3'3'-C T T A A G-5 '3'-C T T A A G-5 '

The high specificity for their recognition site The high specificity for their recognition site means that DNA (target sequence and cloning means that DNA (target sequence and cloning vector) will always be cut reproducibly into vector) will always be cut reproducibly into defined fragments (important for molecular defined fragments (important for molecular cloning)cloning)

Enzyme Organism from which derived Target sequence (cut at *) 5' -3'

Bam HI Bacillus amyloliquefaciens G* G A T C C

Bgl II Bacillus globigii A* G A T C T

Eco RI Escherichia coli RY 13 G* A A T T C

Eco RII Escherichia coli R245 * C C A/T G G

Hae III Haemophilus aegyptius G G * C C

Hind III Haemophilus inflenzae Rd A* A G C T T

Hpa I Haemophilus parainflenzae G T T * A A C

Kpn I Klebsiella pneumoniae G G T A C * C

Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G

SstI Streptomyces stanford G A G C T * C

Sal I Streptomyces albus G G * T C G A C

Taq I Thermophilus aquaticus T * C G A

Xma I Xanthamonas malvacearum C * C C G G G

Note: Only one strand of the target DNA is shown

Frequency of cuttingFrequency of cutting

Because of their restriction site specificity, Because of their restriction site specificity, the restriction endonucleases cut DNA into the restriction endonucleases cut DNA into fragments whose average length is fragments whose average length is determined by determined by the number of base pairs in the restriction site the number of base pairs in the restriction site and to a lesser extent by the ratio of bases in the and to a lesser extent by the ratio of bases in the

DNA. DNA. For DNA that has equal amounts of all four For DNA that has equal amounts of all four

bases, each base has a probabilty of 1/4 at bases, each base has a probabilty of 1/4 at any particular position in the DNA sense any particular position in the DNA sense strand. strand.

Frequency of cuttingFrequency of cutting Because of their restriction site Because of their restriction site

specificity, the restriction endonucleases specificity, the restriction endonucleases cut DNA into fragments whose average cut DNA into fragments whose average length is determined by the number of length is determined by the number of base pairs in the restriction site (and to base pairs in the restriction site (and to a lesser extent by the ratio of bases in a lesser extent by the ratio of bases in the DNA). the DNA).

For DNA that has equal amounts of all For DNA that has equal amounts of all four bases, each base has a probability four bases, each base has a probability of 1/4 at any particular position in the of 1/4 at any particular position in the DNA strand. DNA strand.


For a restriction site of 4 base pairs, For a restriction site of 4 base pairs, the probability of random the probability of random occurrence of that sequence is (1/4)occurrence of that sequence is (1/4)(1/4)(1/4)(1/4) = 1/256. (1/4)(1/4)(1/4) = 1/256.

For 6 base pairs, the probability is For 6 base pairs, the probability is 1/4,096, and for 8 base pairs it is 1/4,096, and for 8 base pairs it is 1/65,536. 1/65,536.


Thus, a restriction endonuclease Thus, a restriction endonuclease with a 6 base pair restriction site with a 6 base pair restriction site would generate fragments whose would generate fragments whose average length is 4,096 base pairs. average length is 4,096 base pairs.

Such fragments are large enough to Such fragments are large enough to contain a complete gene (provided contain a complete gene (provided that the are no cut sites within the that the are no cut sites within the gene for the restriction gene for the restriction endonuclease that is used). endonuclease that is used).


Effect of base composition:Effect of base composition: For DNA whose base composition differs For DNA whose base composition differs

from 50% GT it is necessary to calculate from 50% GT it is necessary to calculate the probability of a site as the product of the probability of a site as the product of the probabilities of each of its components. the probabilities of each of its components.

For example, if a DNA is 66.7% GC (2/3 of For example, if a DNA is 66.7% GC (2/3 of its base pairs are GC) and one assumes its base pairs are GC) and one assumes random orientation of the base pairs, A random orientation of the base pairs, A and T will each have probabilities of 1/6 and T will each have probabilities of 1/6 and G and C will have probabilities of 1/3 and G and C will have probabilities of 1/3 each. each.


Effect of base composition cont’d. :Effect of base composition cont’d. : Thus the probability of GAATTC would be:Thus the probability of GAATTC would be:

(1/3)(1/6)(1/6)(1/6)(1/6)1/3) = 1/11,664(1/3)(1/6)(1/6)(1/6)(1/6)1/3) = 1/11,664

as opposed to 1/4096 when all four as opposed to 1/4096 when all four bases are bases are present in equal amounts. present in equal amounts.

Thus, the average fragment length Thus, the average fragment length generated by generated by Eco Eco RIRI would be longer in a would be longer in a DNA with a higher GC content. DNA with a higher GC content.

Steps in creating Steps in creating recombinant DNArecombinant DNA

The DNA (insert, cloned DNA, target The DNA (insert, cloned DNA, target DNA, foreign DNA) from a DNA, foreign DNA) from a donor donor organism is extractedorganism is extracted

Then DNA is enzymatically Then DNA is enzymatically cleavedcleaved (cut, digested) and (cut, digested) and joinedjoined (ligated) to (ligated) to another DNA entity (cloning vector) another DNA entity (cloning vector) to form a new to form a new recombined DNA recombined DNA molecule molecule (cloning vector-insert DNA (cloning vector-insert DNA construct, DNA construct).construct, DNA construct).

Steps in creating Steps in creating recombinant DNArecombinant DNA

The cloning vector-insert DNA construct is The cloning vector-insert DNA construct is transferred into and maintained within a transferred into and maintained within a host cell, a process called host cell, a process called transformation.transformation.

The host cell that takes up the DNA The host cell that takes up the DNA construct (transformed cells) are construct (transformed cells) are identifiedidentified (by screening) and (by screening) and selectedselected (separated, (separated, isolated) from those that do not.isolated) from those that do not.

Transformation

Many cells lack the ability to take up DNA from their surroundings and are hence not competent

The cell membranes of such cells have to be made porous to make them competent.

Competence can be achieved by treatment of cells with low temperature and calcium chloride.

Heat shock treatment during transformation closes these pores.

Transformation

Transformation is an inefficient process (1 cell in 1000).

Furthermore a few cells will be transformed

by recircularized plasmid DNA

others by nonplasmid DNA

while a few by plasmid-insert DNA construct.

Desirable features of cells used in Transformation

exercises

To ensure that the plasmid-insert DNA construct is perpetuated in its original form the E. coli cells used:

must lack REs

must also be incapable of homologous recombination.

Screening of Transformants After transformation, it is necessary

to identify the cells that contain the plasmid-cloned DNA constructs

This procedure is called screening

The screening method used will depend on the plasmid system used in the transformation process.

Screening when a pBR322 system is used

In a pBR322 system in which the target sequence was inserted into the BamH1 site, the identification is accomplished in two steps:

First the cells from the transformation mixture are plated onto medium that contains ampicillin.

Only those cells that contain either the pBR322 or pBR322-cloned DNA construct (both of which have an intact ampicillin resistance gene) can grow under these conditions.

pBR322

The nontransformed cells are sensitive to ampicillin.

The BamH1 gene of pBR322 is within the tetracycline resistance gene, so insertion of DNA into this gene disrupts the coding sequence and tetracycline resistance is lost.

Cells transformed with the pBR322-cloned DNA construct are resistant to ampicillin but sensitive to tetracycline.

Cells transformed with the recircularized plasmid are resistant to both ampicillin and tetracycline

Hence cells that grow on ampicillin containing medium are transferred to a tetracycline containing medium.

Each location of inoculating cells on a tetracycline-agar plate corresponds to the site of a colony on an original ampicillin-agar plate.

Screening when a pUC 19 vector system is used in

transformation Belongs to the pUC series of plasmids.

They are all based on pBR322 from which 40% of the DNA has been deleted, including the tetracycline gene.

All their cloning sites are also clustered into one site called a multiple cloning site (MCS).

pUC vector contain the pBR322 ampicillin resisitance gene to allow selection for bacteria that have received a copy of the vector.

The MCS lies within the DNA sequence coding for the amino terminal portion of the enzyme β-galactosidase.

The bacteria used with the pUC vectors carry a gene fragment that encodes the carboxyl portion of β-galactosidase.

The two polypeptides by themselves have no activity.

When clones containing the plasmid are plated on medium containing the galactoside, X- gal, colonies with non-recombinant pUC plasmid will turn blue.

β-galactosidase cleaves the X-gal.

This cleavage releases galactose plus an indigo dye that stains the bacterial colony blue.

IPTG is also added to the growth medium to induce the expression of the lacZ gene

Recombinant transformants will produce white colonies on this medium since the galactosidase gene is disrupted by the insert.

Defining a Gene Library

A gene library is a collection of host cells that contain all of the genomic DNA of the source organism.

A gene library is also called a clone bank, or a gene bank.

Creating a gene Library

The source organism’s genomic DNA is digested (cut) into clonable elements and inserted into host cells.

Conditions of the digestion reaction are set to give a partial, not a complete, digestion

Screening a Gene Library After a library is created, the clone (s) (cell

lines) with the target sequence must be identified.

Three popular methods of identification are used:

Colony hybridization with a labeled DNA probe

followed by screening for the probe label

immunological screening for the protein product

screening for protein activity.

Screening by Colony Hybridization

Genomic DNA libraries are often screened by plating out cells from each cell line on to a master plate

Samples of each colony are then transferred to a solid matrix such as a nitrocellulose.

The colonies from the master plate that correspond to samples containing hybridized DNA are then isolated and cultured.

The cells on the membrane are lysed and the target DNA is denatured by alkali treatment

The single strands of DNA are then irreversibly bound to the matrix , this process is often carried out at a high temperature.

Then, the DNA probe, which is labeled with either a radiosotope or another tagging system, is incubated with the bound DNA sample.

If the sequence of nucleotides in the DNA probe is complementary to a nucleotide sequence in the genome of the cell, then base pairing (i.e., hybridization) occurs

The hybridization can be detected by autoradiography or other visualization procedures, depending on the nature of the probe label.

If the nucleotide sequence of the probe does not base pair (bind) with a DNA sequence in the sample, then no hybridization occurs and the assay gives a negative result.

General probes range in length from 100 to more than 1,000 bp, although both larger and smaller probes can be used.

Stable binding requires a greater than 80% match within a segment of 50 bases.

There are at least two possible sources of probes for screening a genomic library:

First, cloned DNA from a closely related organism can be used (a heterologous probe).

Second, a probe can be produced by chemical synthesis.

Probe detection Isotopic detection of hyridization is done

when the probe is labelled with radioactive isotope.

The nitrocellulose membrane with bound DNA hybridized to probe is overlaid with X-Ray film (autoradiography). Darkened areas on the film indicate positions of the DNA probe hybrid.

For nonisotopic detection of hybridization, biotin (or similar nonradioactive label) can be attached to one of the four deoxyribonucleotides that is incorporated into the probe.

Isotopic detection

When a probe with this kind of tag (label) hybridizes to the sample DNA, detection is based on the binding of an intermediary compound (e.g., streptavidin) that carries an appropriate enzyme.

Depending on the assay system, the enzyme can be used for the formation of either a chromogenic (colored) molecule that can be visualized directly or a chemiluminescent response that can be detected by autoradiography

Nonisotopic detection

Analysing hybridization results

Because most libraries are created from partial digestions, a number of colonies (clones) may give a positive response to the probe.

The next task is to determine which clone, if any, contains the complete sequence of the target gene.

Preliminary analyses that use the results of gel electrophoresis and restriction endonuclease mapping reveal the length of each insert and identify those inserts that are the same and those that share overlapping sequences.

If an insert in any one of the clones is large enough to include the full gene, then the complete gene can be recognized after DNA sequencing

(because it will have start and stop codons and a contiguous set of nucleotides that code for the target protein).

If the search for an intact gene fails, then another library can be created with a different restriction endonuclease and screened with either the original probe or probes derived from the first library.

Screening by Immunological Assay

Used if a DNA probe is not available

If a cloned DNA sequence is transcribed and translated, the presence of the protein, or even part of it, can be determined by an immunological assay.

First use a primary antibody that is raised against the protein. Then wash with a secondary antibody.

In many assay systems, the secondary antibody has an enzyme, such as alkaline phosphatase, attached to it.

After washing the matrix, a colorless substrate is added.

If the secondary antibody has bound to the primary antibody, the colorless substrate is hydrolyzed by the attached enzyme and produces a colored compound that accumulates at the site of the reaction.

Cloning DNA Sequences That Encode Eukaryotic Protein in prokaryotic

cells Prokaryotic organisms do not have the

ability to express eukaryotic genes since they do not have the machinery to remove introns.

If Eukaryotic genes are to be expressed in prokaryotic hosts their introns must first be removed.

To achieve this cDNA is made and used for the transformation of prokaryotes

cDNA Synthesis

cDNA synthesis is preceded by extraction of mRNA from the eukaryotic cells

The poly (A) tail of the mRNA can be used to separate the mRNA fraction of a tissue from the ribosomal and transfer RNAs.

mRNA extraction

Extracted cellular eukaryotic RNA is passed through a column packed with cellulose beads to which is bound short chains of thymidine residues, each about 15 nucleotides long (oligo-dT15).

The poly (A) tails of the messenger RNA molecules bind by base pairing to the oligo-dT chains.

cDNA Synthesis The process is divided into two steps:1. First Strand Synthesis

After the mRNA fraction is purified the following are added:

i. short (unbound) sequences of oligo-dT molecules

ii. the enzyme reverse transcriptase iii. the four deoxyribonucleotides (dATP,

dTTP, dGTP, dCTP).

The oligo-dT molecules base pair with the poly (A) tail regions and provide an available 3’ hydroxyl group to prime the synthesis of a DNA strand.

2. Second Strand Synthesis

The second DNA strand is synthesized by the addition of the Klenow fragment of E. coli DNA polymerase I:

The polymerase uses the first DNA strand as a template and adds deoxyribonucleotides to the growing strand, starting from the end of the hairpin loop.

After the reaction is complete, the sample is treated

with:

the enzyme Rnase H, which degrades the mRNA molecules and

S1 nuclease, which opens the hairpin loops and degrades single-stranded complementary DNA (cDNA) copies of the more prevalent mRNAs in the original sample.