1. DNA is Composed of Complementary Strands
Last time we started the discussion of DNA – deoxyribonucleic acids. DNA has three major functions in a cell: serve as a repository of genetic information; replicate during cell division to provide a copy of genome to daughter cells, and serve as a template for gene expression (RNA and protein synthesis)
The secondary structure of DNA was first determined by Watson and Crick. This is the major structure of DNA found in chromosomes – also called B-DNA.
As a reminder, the major features of this structure were:
Two polynucleotide chains running in the opposite directions coil around a common axis to form a right handed helix (explain).
2. The planes of the bases are perpendicular to the helix axis, while the sugars are nearly parallel to the axis. The two chains are held together by stacking (hydrophobic) interactions, hydrogen bonds, and electrostatic forces.
3. Each base is paired with the complementary base in the opposite strand by specific hydrogen bonds. Complementary sequences – redundant information Important for maintaining genetic stability and for replication.
4. The helical axis passes through the middle of each base pair.

2. Base Pairing is Determined by Hydrogen Bonding
Base pairing is in the order A:T, which contains two hydrogen bonds, and G:C, which contains three hydrogen bonds.
Only two types of base pairs can be accommodated without altering the positions of the sugars.
same size

3. Forces stabilizing DNA double helix
Hydrogen bonding (2-3 kcal/mol per base pair)
Stacking (hydrophobic) interactions
(4-15 kcal/mol per base pair)
3. Electrostatic forces.
Cooperative forces: each contributes a little, but adds up because DNA chains can be millions od nculeotides long.

4. B-DNA •Sugars are in the 2’ endo conformation.
•Bases are the anti conformation.
•Bases have a helical twist of 36º
(10.4 bases per helix turn)
Helical pitch = 34 A
23.7 A
right handed helix
helical axis passes through
base pairs
7.0 A
planes of bases are nearly
perpendicular to the helix axis.
3.4 A rise between base pairs
Wide and deep
More recent studies used X-ray crystallography and NMR to confirm the original structure by Watson and Crick. Right handed helix- winds about the same direction in which fingers of a right hand curl when the thumb is pointing upward.
Helical axis passes through base pairs
•Contains a minor and a major groove that wind about the outside of the helix
• Helical structure repeats every 34 A
Narrow and deep

5. DNA can deviate from Ideal Watson-Crick structure
Helical twist ranges from 28 to 42°
Propeller twisting 10 to 20°
Base pair roll
These studies also have shown significant local deviations of the DNA structure from the classical WC structure:
Propeller twisting maximize stacking

6. Major and minor groove of the double helix2 N N NH N N N
C-1’ NH 2 O
C-1’ HN N O NH 2
C-1’
Wide and deep
Major and minor groove are the two surfaces that wind about the outside of the helix. They are formed by the edges of the stacked bases. These grooves are distinct because they have different H-bonding patterns and different size.
Narrow and deep

7. Major groove and Minor groove of DNA
Hypothetical situation: the two grooves would have similar size if dR residues
were attached at 180° to each other
To deoxyribose-C1’
C1’ -To deoxyribose
The grooves are formed by the edges of stacked bases and have different sizes because deoxyribose residues are attached asymmetrically (not 180 degrees). The minor groove is the one in which C-1’ –helix axis’c1’ angle is less than 180 degrees (recall that the helix axis passes trough the middle of each base pair in B-DNA) and the major groove is on the opposite side of each base pair. HN N O NH 2
C-1’ NH N O 2 H
C-1’
C-1’

8. B-type duplex is not possible for RNA
Although B-DNA solvbed by W and C is the major form of DNA in chromosomes, other types of secondary structure are possible. For example, duplexes containing RNA cannot form B type duplex because of the steric clash between its 2’-OH and the phosphodiester. Instead, it forms an A-type duplex.
steric “clash”

9. A-form helix: dehydrated DNA; RNA-DNA hybrids
•Sugars are in the 3’ endo conformation.
•Bases are the anti conformation.
•11 bases per helix turn
Helical pitch = 25.3 A
Right handed helix
planes of bases are tilted
20 ° relative the helix axis.
2.3 A rise between base pairs
Like B-DNA, it is a right handed duplex. Unlike B-DNA, bases in A-DNA are not perpendicular to helical axis (20 degree tilt). Wider and flatter than the B-duplex, contains an axial hole.
25.5 A
Top View

10. The sugar puckering in A-DNA is 3’-endo

11. Living Figure – A-DNA http://bcs.whfreeman.com/biochem5
Looks like a flat ribbon wound about a 6A in diameter cylindrical hole.
Flat ribbon wound around a 6 A in diameter cylindrical hole

12. A-DNA has a shallow minor groove and a deep major groove• •
Helix axis
B-DNA
A-DNA

13. Z-form double helix: polynucleotides of alternating purines and pyrimidines (GCGCGCGC) at high salt
• Backbone zig-zags because sugar puckers alternate between 2’ endo pyrimidines and 3’ endo (purines)
• Bases alternate between anti (pyrimidines) and syn conformation (purines).
•12 bases per helix turn
Helical pitch = 45.6 A
Left handed helix
planes of the bases are
tilted 9° relative the helix
axis.
3.8 A rise between base pairs
Repeated GC sequence
• Flat major groove
• Narrow and deep minor groove
18.4 A

14. Sugar and base conformations in Z-DNA alternate:
5’-GCGCGCGCGCGCG
3’-CGCGCGCGCGCGC
C: sugar is 2’-endo, base is anti
G: sugar is 3’-endo, base is syn

15. Living Figure – Z-DNA

17. Biological relevance of the minor types of DNA secondary structure
Although the majority of chromosomal DNA is in B-form,
some regions assume A- or Z-like structure
Runs of multiple Gs are A-like
The upstream sequences of some genes contain
5-methylcytosine = Z-like duplex
Reversible conversion to m
Structural variations play a role in DNA-protein interactions
RNA-DNA hybrids and ds RNA have an A-type structure

18. Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins
The edges of base pairs displayed to DNA major and minor groove
contain potential H-bond donors and acceptors: N H 2 N O H 2 N n h o h
The edges of the DNA bases displayed on different sides of the AT and GC base pairs in DNA duplex are exposed to the major and the minor groove. The two grooves are structurally distinct are lined by potential hydrogen bond donors and acceptors. Proteins can bind to the major and minor groove of the double helix in a sequence-specific manner by forming new H-bonds. The key sequence recognition features are governed by the hydrogen bonding patterns:
n= Nitrogen hydrogen bond acceptor
o= Oxygen hydrogen bond acceptor
h= Amino hydrogen bond donor

19. Hydrogen bond donors and acceptors on each edge of a base pair

20. Structural characteristics of DNA facilitating DNA-Protein Recogtnition
Major and major groove of DNA contain sequence-
dependent patterns of H-bond donors and acceptors.
Sequence-dependent duplex structure (A, B, Z, bent
DNA).
Hydrophobic interactions via intercalation.
Ionic interactions with phosphates.

21. Groove binding drugs and proteins
Leucine zipper proteins bind DNA
major groove
5’-ATT-3’
4,6-diamidino-2-phenylindole (DAPI) – synthetic antibiotic interferes with DNA-processing enzymes (regulatory proteins). High affinity binding to the minor groove in AT sequences. (5’-ATT). N-H of indole forms an H-bond with the O-2 of T. Inhibits gene expression in bacteria by inhibiting binding of RNA Pol II to its consensus sequence (TATA box) – inhibits initiation of gene expression
Other minor groove bionders: netropsin and distamycin
The interaction between proteins and DNA is important in such cellular events as DNA packing, DNA replication, DNA transcription, and gene regulation. The regulation of gene expression is crucial for the healthy living of any organism. The cell needs to be able to respond to the constantly changing environment. he leucine zipper is an interesting structure made up of two a-helical segments of protein that have leucines facing each other along the length of the helices, allowing them to dimerize and form a symmetric interface that can bind to the DNA on both sides of the double helix (see figure). The leucine zipper motif will be illustrated with the GCN4 (protein)-AP1 (DNA) complex, a protein involved in activating transcription in yeast.
The phosphodiester backbone of the bound DNA strand is negatively charged and able to interact with the basic region of the leucine zipper, which is positively charged. Notice how well the DNA and protein fit together. The general interactions that take place between the protein and DNA are the nonspecific charge interactions between the phosphodiester backbone of the DNA and the basic amino acids of the leucine zipper. This associates the DNA
and protein together.
Others: netropsin, distamycin,
Hoechst 33258

22. Triple helix and Antigene approach
Triple helix may involve DNA and RNA strands. Can form in vivo. Potential to target specific genes to regulate their expression. Synthetic 17-30mers that bind selectively to specific DNA sequences can inhibit transcription. In these structures the 3rd helix occupies the major groove binding purine rich seqeunces by specific H-bonds. Hoogsteen base pairing is a Non-traditional base pairing – involves the N-7 position of guanine. Restricted to homopurine seqeunces.
Hoogsteen base pairing = parallel
Reversed Hoogsteen = antiparallel

23. Biophysical properties of DNA
Facile denaturation (melting) and re-association of the duplex
are important for DNA’s biological functions.
In the laboratory, melting can be induced by heating.
Single strands T°
duplex
The non-covalent bonds that stabilize the DNA double helix can be disrupted by heating or by reducing salt concentration. The process of strand separation is called denaturation or melting.
Denaturation of DNA occurs over a narrow temperature range and results in changes in many of its physical properties. Experimentally can follow by measuring UV absorbance as a function of temperature. DNA denaturation is a cooperative process that occurs over an narrow temp range. This indicates that destabilization of one part of the structure affects the stability of the other. The mid point of the melting curve (melting temperature) corresponds to DNA in which 50% of the duplex is denatured. The higher Tm the more stable is the duplex. DNA melting is reversible – DNA strands will re-anneal when the temperatire is lowered below Tm. Can also form DNA-RNA duplexes. This is the basis for many molecular biology techniques
Experimentally can follow by measuring UV absorbance as a function of temperature. DNA denaturation is a cooperative process that occurs over an narrow temp range. mThis indicates that destabilization of one part of the structure affects the stability of the other. The mid point of the melting curve (melting temperature) corresponds to DNA in which 50% of the duplex is denatured. The higher Tm the more stable is the duplex. DNA melting is reversible – DNA strands will re-anneal when the temperatire is lowered below Tm. Can also form DNA-RNA duplexes. This is the basis for many molecular biology techniques
Hybridization techniques are based on the affinity of complementary
DNA strands for each other.
Duplex stability is affected by DNA length, % GC base pairs, ionic strength, the presence of organic solvents, pH
Negative charge – can be separated by gel electrophoresis

24. Separation of DNA fragments by gel electrophoresis
Polyacrylamide gel:
DNA strands are negatively charged –
migrate towards the anode
Migration time ~ ln (number of base
pairs)
DNA molecules are negatively charged due to the presence of ionized phosphate groups. They can be separated by size using polyacrylamide gel electrophoresis. In the presence of radicals, acrylamide/bis-acrylamide mixture polymerizes producing a porous gel matrix. In the presence of electric field, DNA molecules will migrate towards the positive electrode (anode), with the migration time proportional to their size.
DNA molecules can be visualized on the gel by staining with fluorescent dyes or by autoradiography.

25. DNA Topology DNA has to be coiled to fit inside the cell
Organism
Number of base pairs
Contour length, m
E. Coli
bacteria
4,600,000
1,360
SV40 virus
5,100
1.7
Human chromosomes
48,000,000-
240,000,000
1.6 – 8.2 cm
The length of DNA molecule is considerably greater than the dimension of the cell. DNA double helix does not exist in a cell as a long straight rod: it is coiled (folded back) n space to fit the dimensions of the cell. This phenomenon is called DNA supercoiling. For example,  phase (a virus) contains 4.8 x 104 bp of DNA (15.5 m long) but the viral particle is only 0.19 m long. Human cell contains 6 x 109 nucleotides of chromosomal DNA packaged in 46 chromosomes (total length = 2 meters), but it all fits inside the nucleus (diameter = 10 m). In both prokaryotes and eukaryotes, DNA must be tightly packaged.
DNA polymers must be folded to fit into the cell or nucleus (tertiary structure).

26. DNA Topology:
Negative supercoiling: double helix is twisted in the direction opposite to the direction of the helix (underwound DNA). Negative supercoiling generates a torsional force that helps unwind DNA when required for replication and transcription.
Positive supercoiling: double helix is twisted in the same direction as the winding of the helix (overwound DNA).

27. DNA Topology: linking number
DNA conformations differing in their degree of supercoiling (topoisomers) are defined by their linking number. Linking number (Lk) is the total number of times one strand winds around the other in the right-handed direction. Lk is the topological property of DNA moilecule and cannot be changed without cleaving one or both DNA strands.
• Tw (twisting number) is the number of turns of the helix. T = # of base pairs/#bp per turn
In B-DNA, there are 10.4 #bp per turn, T = # of base pairs/10.4
• Wr (writhing number) is number of superhelical turns
For a closed circle of DNA or DNA with fixed ends, Lk is constant and cannot be changed without breaking DNA stands. Therefore, if Wr is changed due to positive or negative supercoiling, Tw has to change in the opposite direction
If Lk = const., ΔTw = - ΔWr
 If Lk is changed, it is more energetically favorable to change Wr, rather than Tw: ΔLk = ΔWr + ΔTw

28. Consider a 260 bp B-duplex:
Note that supercoiling is only possible for DNA melecules without free ends (e.g. curcular DNA OR DNA with fixed ends)

29. Connect the ends to make a circular DNA:
Tw = 260/10.4 = 25
 If we simply connect the ends of a linear 260 bp duplex:
No supercoiling
Tw = 260/10.4 = 25, Wr = 0 , Lk = = Floppy

31. If we disconnect the circle and unwind DNA helix by two turns, then re-connect the ends (D):
Lk = 23, Tw = 23, Wr = 0

32. Since the underwound conformation (D) is strained due to the loss of stacking interactions, the entire duplex spontaneously twists in the opposite direction to give a structure with two superhelical turns (E) and restoring base stacking (negative supercoilng):
•Lk = 23, Tw = 25, Wr = -2
This DNA is topologically identical to relaxed unwound DNA in previous figure but it geometrically different: densely packed and compact – base stacking is maintained, more energetically favorable than D.

33. An electron micrograph of negatively supercoiled and relaxed DNA
Functions of DNA supercoiling:
Stryer Fig

34. Organization of chromosomal DNA
• Chromosomal DNA is organized in loops (no free ends)
• It is negatively supercoiled: 1 (-) supercoil per 200 nucleotides
145 bp duplex
Histone octamer (H2A, H2B, H3, H4)2
H1 is bound to the linker region
Chromosomal DNA is linear and is organized in loops and supelcoiled by wrapping around small basic proteins called histones. There are five types of histones: H1, H2A, H2B, H3, and H bases of DNA duplex writhed around each histone octamer (containing two copies of each H2A, H2B, H3, H4) constitutes nucleosomal core. Nucleosomal cores are separated by linker DNA loosely associated with histone H1. Nucleosomes are further packed into a higher order structure. DNA + histones = chromatin. Negative supercoiling generated by writhing of DNA duplex around histones ( 1 (-) supercoil per 200 nucleotides) is useful for processes requiring DNA strand separation, such as replication, recombination and transcription.

35. Enzymes that control DNA supercoiling: DNA Topoisomerases
Change the linking number (Lk) of DNA duplex by
concerted breakage and re-joining DNA strands
Topoisomerase enzymes
Topoisomerases I
Relax DNA supercoiling by
increments of 1 (cleave one strand)
Topoisomerases II
Change DNA supercoiling by
the increments of 2
(break both strands)
Usually introduce negative supercoiling
Topoisomerases change the linking number of DNA duplex by catalyzing the concerted breakage and re-joining of DNA strands.
Two general classes: Topomerases I (cleave one DNA strand) and Topoisomerases II (cleave both DNA strands).

36. Human DNA Topoisomerase I: DNA: side view
20Å
Topoisomerase I enzymes relax supercoiled DNA by changing the linking number by the increments of 1. This reduces the torsional stress, facilitating DNA replication, repair, and transcription.
• use the energy stored in supercoiled double-stranded DNA
• cleave one of the strands, forming a reversible covalent complex and allowing for relaxation of torsional strain
• can act repeatedly to reduce supercoiling in steps of 1
Stryer Fig

37. Mechanism of DNA Topoisomerases IOH
723
P-Topo
1)      Cut one DNA strand, generating a transient strand break. The 5’ phosphate is covalently linked to a Tyr residue of the enzyme (Tyr 723 in the human enzyme)
2) Pass the intact strand through the gap.
3) 3’ OH on the other end of DNA chain attacks the enzyme-DNA intermediate restoring the original DNA chain and releasing the Tyr:
The enzyme cuts one DNA strand and forms a covalent bond betweewn a Tyr residue and the 5’ phosphate
The intact strand is passed through the resulting gap
 Wr = 1

38. Drugs that inhibit DNA Topoisomerase I
Camptothecin, Topotecan and analogs block eukaryotic topoisomerase I. These drugs binds to and stabilize the DNA-topoisomerase complex. This prevents DNA strand re-ligation. The collision of DNA replication fork with the cleaved strand leads to DNA degradation, inhibition of replication, and cell cycle arrest.
Topotecan is used in the clinic for the treatment of colorectal, ovarian, and small cell lung tumors.
• Camptothecin, topotecan and analogs
• Antitumor activity correlates with interference with topoisomerase activity
• Stabilizes topoisomerase I-DNA intermediate, preventing DNA strand re-ligation
Used in treatment of colorectal, ovarian, and small cell lung tumors

39. Enzymes that control DNA supercoiling: DNA Topoisomerases
Change the linking number (Lk) of DNA duplex by
concerted breakage and re-joining DNA strands
Topoisomerase enzymes
Topoisomerases I
Relax DNA supercoiling by
increments of 1 (cleave one strand)
Topoisomerases II
Change DNA supercoiling by
the increments of 2
(break both strands)
Usually introduce negative supercoiling

40. Topoisomerases II
• Most of Topoisomerases II introduce negative supercoils (e.g. E. coli DNA Gyrase)
• Require energy (ATP)
• Each round introduces two supercoils ( Wr = - 2)
• Necessary for DNA synthesis
• Form a covalent DNA-protein complex similar to Topoisomerases I
Topoisomerases II : change the linking number by the increments of 2, usually increasing (-supercoiling
• require energy (ATP)
• only act on double stranded not single stranded DNA
• each round introduces two negative supercoils (  Wr = -2)
• can act repeatedly to introduce or relax more supercoiling

41. Yeast DNA Topoisomerase II
Yeast Topo II: heart-shaped dimer with a large central cavity. Cavity has gates on the top and bottom essential for topo action.
Green: ATP binding domain
Yellow: carboxy-terminal domain
Stryer Fig

42. Topoisomerase II - mechanism
One portion of Dna duplex binds Topo II binding site includes Tyrosine residues involved in catalysis.
Each monomer bind a molecule of ATP and and another portion of the duplex (T). ATP binding induces a conformational change, causing the two ATP-binding domains to come together, trapping the T segment.
Both strands of the G duplex are cleaved, and the 5’-phosphate ends are covalently bound to the enzyme through tyrosine-phosphodiester linkage.
T duplex passes through the “gate” in the G duplex into the central cavity of the enzyme.
G duplex strands are sealed, and the T segment exits through the bottom of the enzyme
ATP hydrolysis induces conformational change leading to separation of ATP-binding domains. Noe the enzyme is ready to bind another T segment
Stryer Fig

43. Drugs that inhibit bacterial Topoisomerase II (DNA gyrase)
Interfere with breakage and rejoining DNA ends:
Inhibit ATP binding:
Bacterial Topoisomerases II (Gyrases) are useful targets for antibiotics, since negative supercoiling is necessary for the replication of bacteria. By selectively inhibiting gyrase, these drugs stop cell division, inhibiting bacterial growth.
- Novobiocin = blocks ATP binding to gyrase – only 1 round of supercoiling is possible
- Ciprofloxacin and and Nalidixic Acid = inhibit cleavage and re-ligation of DNA strands. Stabilize DNA-protein complex (cleavable complex)

44. Enzymes that cut DNA: exonucleases5’ A OH HO 5’ 3’ 3’ 5’
+ dNMPs
• Degrade DNA in a stepwise manner by removing deoxynucleotides in 5’  3’ (A) or 3’  5’ direction (B)
• Require a free OH
• Most exonucleases are active on both single- and double-stranded DNA
• Used for degrading foreign DNA and in proofreading during DNA synthesis
Exonucleases: cleave nucleotides one at a time from an end of a polynucleotide chain; may be specific for 5’ end or the 3’ end of DNA chain, thus the name 5’3’ and 3’5’ exonucleases. Examples: snake venom phosphodiesterase (3’5’), calf intestinal phosphodiesterase (5’3’)
• Degrade DNA in a stepwise manner by removing mononucleotides in
5’ to 3’ (A) or 3’ to 5’ direction (B)
• Require a free OH
• Most exonucleases are active on both single- and double-stranded DNA
• Used for degrading foreign DNA and in proofreading during DNA synthesis B HO H 3’
Nucleobase
Phosphate group
2’-deoxyribose

45. DNA Endonucleases
• Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends 5’
3’-OH
5’-P
5’-P
3’-OH
• some endonucleases cleave randomly (DNase I, II)
• Type II Restriction endonucleases are highly sequence specific
EcoRI recognition site:
Endonucleases: enzymes that cleave internal phosphodiester bonds within DNA chain resulting in 3’-OH and 5’-phosphate ends
 Type II restriction endonucleases (RE) cut DNA at specific sites defined by a palindromic (inverted repeat) DNA sequence.
• RE originate from bacteria – used to degrade foreign DNA
• RE recognize unmethylated DNA (their own DNA is methylated)
• RE cut DNA at palindromic (two fold rotational axis of symmetry) recognition sites. The cuts are symmetrically positioned and can be staggered or even.
Palindromic site
(inverted repeat)
• RE are found in bacteria where they are used for protection against foreign DNA

46. Recognition sequences of some common restriction endonucleases
Because of their ability to fragment DNA, restriction endonucleases are important tools used by molecular biology for DNA manipulation. Incubation of DNA with RE generates a unique set of fragments, often with “sticky ends”. This can be used for pricise cutting and re-ligation of DNA in DNA cloning.
Cut DNA at
Staggered cut: short ss tail at two ends of ea fragment – cohesive ends

47. DNA Restriction Enzyme EcoR V
enzyme has same two fold axis of symmetry
• highly specific; recognition takes place in the major groove of DNA via hydrogen bond formation between amino acids of the enzyme (Arg, Glu) and DNA nucleobases

48. Applications of Restriction Endonucleases in Molecular Biology
DNA fingerprinting (restriction fragment length polymorphism).
2. Molecular cloning (isolation and amplification of genes).
Applications of restriction endonucleases:
Restriction fragment polymorphism (RFPM) to determine whether two DNA samples are from the same source (e.g. forensic DNA sample from a suspect and DNA recovered from the evidence/crime scene) and to diagnose genetic diseases.
Molecular cloning (see below).
RFPM analysis involves cutting a region of DNA with restriction enzymes and separation of the resulting fragments on a gel. Since human genomes differ by about 1 base per 1000 nucleotides, these variations in DNA sequence eliminate or create new RE recognition sites, resulting in characteristic patterns of fragments. These cleavage patterns can be compared to establish whether the DNA is from the same individual and to diagnose rare genetic diseases.

49. Southern blotting
Hybridization techniques – make it possible to find a specific DNA sequence by its ability to bind complementary NA sequence

50. Restriction fragment length polymorphisms are used to compare DNA from different sources
Fragment size differences detected by Southern blotting with a dNA sequence complementary to a specific highly variable region of human genome

51. • Forms phosphodiester bonds between 3’ OH and 5’ phosphate
DNA Ligase
AMP + PPi O P O -O O- O P O OH
DNA Ligase +
(ATP or NAD+) O-
• Forms phosphodiester bonds between 3’ OH and 5’ phosphate
• Requires double-stranded DNA
• Activates 5’phosphate to nucleophilic attack by transesterification
with activated AMP
Enzymes that join (ligate) DNA fragments are called DNA Ligases
• catalyze the formation of phosphodiester bond between 3'- OH and 5'- phosphate
 require energy: bacteria use NAD+ and animal cells use ATP
• only acts on double stranded not single stranded DNA
• needed for DNA repair, DNA synthesis (see next chapters)

52. DNA Cloning: recombinant DNA technology
DNA fragments can be precisely cut with RE and joined by DNA ligase Molecular biologists create large DNA molecules with special characteristics by cleaving and joining DNA fragments. These DNA constructs can be then used to amplify DNA for sequencing and other analyses or to produce recombinant proteins.
DNA cloning procedure
1.      Isolate DNA
2.      Cut with specific Restriction Endonucleases (RE)
3.      Purify by gel
4.      Cloning vectorDNA of interestInsert into a cloning vector (e.g. circular plasmid). The cloning vector is cut with the same RE to generate complementary single stranded ends. Ligate:
Cleave DNA at specific sites witrh restriction endonucleases
Insert in a plasmid, amplify to generate billions of copies
1.      Introduce in bacteria.
2.      Grow bacteria.
3.      Isolate DNA or expressed protein.

53. Human Genetic Polymorphisms
Human genome size: 3.2 x 109 base pairs
30,000 genes
2-4 % of total sequence codes for proteins
Human genetic variation:
 1 sigle nucleotide polymorphism (SNP) per 1,300 bp
Major findings: 1) there are only 30,000 genes in human DNA (expected 120,000 or more).
2) only 2-4% of the human genome codes for proteins
3) many repeating sequences of unknown function
Major implications for pharmacy: 1) further understanding of the molecular basis of disease
2) individualized therapies (designer drugs)
3) potential new drug targets

54. Examples of genetic polymorphisms of drug metabolizing enzymes
 Enzyme substrate examples DNA regions involved
cytochrome 2B6 cyclophosphamide exons 1,4,5, and 9
tamoxifen
benzodiazepines
cytochrome 2D6 debrisoquine internal base changes
cytochrome 1A2 caffein 5' flanking region
phenacetin
N-acetyltransferase aromatic amines
Interindividual differences in DNA sequence may include genetic polymorphisms in genes coding for drug-metabolizing enzymes (e.g. cytochrome P450 monooxygenases) can lead to alterations in enzyme function. The lack of activity of the enzymes involved in clearing a drug from the body (poor metabolizers) can result in exaggerated clinical response and more severe side effects, while fast metabolizers require higher amounts of the drug to achieve a therapeutic effect. Several of these genes are known to be highly variable, especially between ethnic groups.

55. DNA Structure: Take Home Message
Genetic information is stored in DNA.
DNA is a double stranded biopolymer containing repeating units of nitrogen base, deoxyribose sugar, and phosphate.
DNA can be arranged in 3 types of duplexes which contain major and minor grooves.
DNA can adopt several topological forms.
There are enzymes that will cut DNA, ligate DNA, and change the topology of DNA.
Human genome contains about 3.2 billion base pairs. Inter-individual differences are observed at about 1 per 1,000 nucleotides.