1. Recombinant DNA Technology
UNIT 5
Recombinant DNA Technology

2. Objectives Define Recombinant DNA technology
Outline steps involved in creating recombinant DNA
Define type II restriction enzymes and their use in recombinant DNA technology
Explain restriction mapping and construct restriction maps
State the properties of cloning vectors and reproduce genetic map of common cloning vectors
Outline the steps involved in cloning prokaryotic DNA into a plasmid cloning vector
Outline the steps involved in cloning eukaryotic DNA into a plasmid cloning vector
Understand the methods of transformation and the steps involved in the selection of transformants
Explain the concept of gene libraries and methods of screening a gene library

3. Recombinant DNA technology
a body of techniques for cutting and splicing together different pieces of DNA.
When segments of foreign DNA are transferred into another cell or organism, the protein for which they code may be produced along with substances coded for by the native genetic material of the cell or organism.
These cells become "factories" for the production of the protein coded for by the inserted DNA

4. Restriction Enzymes
The major tools of recombinant DNA technology are restriction enzymes (nucleases)
first discovered in the late 1960s
They work by cutting up the foreign DNA, a process called restriction.
Most restriction enzymes are very specific
recognizing short, specific nucleotide sequences in DNA molecules and cutting at specific points within these sequences.
There are hundreds of restriction enzymes and more than 150 different recognition sequences.

5. Restriction enzymes
Nucleases are further described by addition of the prefix "endo" or "exo" to the name:
endonuclease applies to sequence specific nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule.
exonucleases function by removing nucleotides from the ends of the molecule

6. Types of Restriction Enzymes
Type I  Recognise specific sequences·but then track along DNA (~ bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA
Type II  Recognise a specific target sequence in DNA, and then break the DNA (both strands), within the recognition site
Type III  Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site

7. Restriction Endonucleases
The first endonucleases discovered was from Escherichia coli EcoRI
A restriction endonuclease functions by "scanning" the length of a DNA molecule.
Once it encounters its particular specific sequence (recognition site), it will bond to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones of the double helix.
Once the cuts have been made, the DNA molecule will break into fragments.

8. Restriction Endonucleases
The positions of these two cuts, both in relation to each other, and to the recognition sequence itself, are determined by the identity of the restriction endonuclease
The length of the recognition site for different enzymes can be four, five, six, eight or more nucleotide pairs long.
Restriction endonucleases that cleave recognition sites of four and six nucleotide pairs are used for most of the protocols for molecular cloning since these restriction sites are common in DNA.

9. Restriction Endonucleases
Different endonucleases yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way.
Some restriction endonucleases digest DNA leaving 5′-phosphate extensions(protruding ends, sticky ends)
Some leave 3′-hydroxyl extensions
Some cut the backbone of both strands within a recognition site to produce blunt-ended (flush-ended) DNA molecules.

10. Use of Restriction Enzymes in recombinant DNA Technology

11. How Restriction Enzymes are named
generally have names that reflect their origin—
The first letter of the name (capitalized ) comes from the genus of the bacteria
the second two letters (lower case) come from the species
Roman numerals following the nuclease names indicate the order in which the enzymes were isolated from that strain of bacteria.
For example EcoRI comes from Escherichia coli RY13

12. Type II Restriction Enzymes
Many Type II restriction endonucleases recognise PALINDROMIC sequences
Symmetrical sequences which read in the same order of nucleotide bases on each strand of DNA (always read 5' to 3')
For example, EcoRI recognises the sequence
5'-G A A T T C-3'
3'-C T T A A G-5 '
The high specificity for their recognition site means that DNA (target sequence and cloning vector) will always be cut reproducibly into defined fragments (important for molecular cloning)

13. Organism from which derived Target sequence (cut at *) 5' -3' Bam HI
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

14. Frequency of cutting
Because of their restriction site specificity, the restriction endonucleases cut DNA into fragments whose average length is determined by
the number of base pairs in the restriction site
and to a lesser extent by the ratio of bases in the DNA.
For DNA that has equal amounts of all four bases, each base has a probabilty of 1/4 at any particular position in the DNA sense strand.

15. Frequency of cutting
Because of their restriction site specificity, the restriction endonucleases cut DNA into fragments whose average length is determined by the number of base pairs in the restriction site (and to a lesser extent by the ratio of bases in the DNA).
For DNA that has equal amounts of all four bases, each base has a probability of 1/4 at any particular position in the DNA strand.

16. Frequency of cutting
For a restriction site of 4 base pairs, the probability of random occurrence of that sequence is (1/4)(1/4)(1/4)(1/4) = 1/256.
For 6 base pairs, the probability is 1/4,096, and for 8 base pairs it is 1/65,536.

17. Frequency of cutting
Thus, a restriction endonuclease with a 6 base pair restriction site would generate fragments whose average length is 4,096 base pairs.
Such fragments are large enough to contain a complete gene (provided that the are no cut sites within the gene for the restriction endonuclease that is used).

18. Frequency of cutting Effect of base composition:
For DNA whose base composition differs from 50% GT it is necessary to calculate the probability of a site as the product of the probabilities of each of its components.
For example, if a DNA is 66.7% GC (2/3 of its base pairs are GC) and one assumes random orientation of the base pairs, A and T will each have probabilities of 1/6 and G and C will have probabilities of 1/3 each.

19. Frequency of cutting Effect of base composition cont’d. :
Thus the probability of GAATTC would be:
(1/3)(1/6)(1/6)(1/6)(1/6)1/3) = 1/11,664
as opposed to 1/4096 when all four bases are present in equal amounts.
Thus, the average fragment length generated by Eco RI would be longer in a DNA with a higher GC content.

20. Steps in creating recombinant DNA
The DNA (insert, cloned DNA, target DNA, foreign DNA) from a donor organism is extracted
Then DNA is enzymatically cleaved (cut, digested) and joined (ligated) to another DNA entity (cloning vector) to form a new recombined DNA molecule (cloning vector-insert DNA construct, DNA construct).

21. Steps in creating recombinant DNA
The cloning vector-insert DNA construct is transferred into and maintained within a host cell, a process called transformation.
The host cell that takes up the DNA construct (transformed cells) are identified (by screening) and selected (separated, isolated) from those that do not.

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

28. pBR322

29. 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.

30. 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.

31. 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.

33. 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.

34. X- gal, colonies with non-recombinant pUC plasmid will turn blue.
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.

35. 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.

36. 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.

37. 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

38. 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.

39. 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.

41. 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

42. 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.

43. 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.

44. 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.

45. Isotopic detection

46. 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

47. Nonisotopic detection

48. 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.

49. 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).

50. 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.

51. 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.

52. 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.

54. 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

55. 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.

56. 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.

57. 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.

59. 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.

61. 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.