The document outlines an introduction to cloning and recombinant technology. It discusses DNA cloning, DNA sequencing, detection of disease genes, and polymerase chain reaction (PCR). Specifically, it covers DNA cloning techniques using restriction enzymes and ligases, DNA sequencing methods using dideoxynucleotides, detecting disease genes via Southern blotting and restriction fragment length polymorphisms, and applications of PCR in medicine and forensics such as diagnosing genetic diseases and identifying pathogens.

1. Introduction to Cloning and Recombinant
Technology: Lecture Outline
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics

2. Introduction to Cloning and Recombinant
Technology: Lecture Outline
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics

3. Plasmid
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from
1,000 to 8,000 kilobase pairs.
Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule.
Bacterial Genome: The collection of all of the genes present on the bacteria’s
chromosome or its extrachromosomal genetic elements.
DNA is the genetic material of most
organisms (from bacteria to humans)

4. Basics: Nucleotides are the
building blocks of DNA
Only in RNA,
not DNA

5. Deoxyribonucleic acid (DNA) is a long
double-stranded chain of nucleotides
• DNA is the hereditary material
passed on from generation to
generation.
• DNA is made up of four
nucleotides: A, C, G, and T.
• A always pairs with T.
• C always pairs with G.
• The two strands of DNA are in an
antiparallel configuration.
• Two complementary DNA strands
will separate when heated, and
will spontaneously pair together
again (hybridize) when cooled.

6. DNA Double Helix

7. Introduction to Cloning and Recombinant
Technology: Lecture Outline
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics

8. What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an
original molecule or cell
• To "clone a gene" is to make many copies of it - for
example, by replicating it in a culture of bacteria.
• Cloned gene can be a normal copy of a gene (= “wild
type”).
• Cloned gene can be an altered version of a gene (=
“mutant”).
• Recombinant DNA technology makes manipulating genes
possible.

9. Restriction Enzymes
• Bacteria have learned to "restrict" the possibility of
attack from foreign DNA by means of "restriction
enzymes”.
• Cut up “foreign” DNA that invades the cell.
• Type II and III restriction enzymes cleave DNA chains
at selected sites.
• Enzymes may recognize 4, 6 or more bases in
selecting sites for cleavage.
• An enzyme that recognizes a 6-base sequence is
called a "six-base cutter”.

10. Basics of type II Restriction Enzymes
• No ATP requirement.
• Recognition sites in double stranded DNA have a 2-fold
axis of symmetry – a “palindrome”.
• Cleavage can leave staggered or "sticky" ends or can
produce "blunt” ends.

11. Recognition/Cleavage Sites of Type II
Restriction Enzymes
Cuts usually occurs at
a palindromic sequence
SmaI: produces blunt ends
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends
5´ GAATTC 3´
3´ CTTAAG 5´
Examples of Palindromes:
Don't nod
Dogma: I am God
Never odd or even
Too bad – I hid a boot
Rats live on no evil star
No trace; not one carton
Was it Eliot's toilet I saw?
Murder for a jar of red rum
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Go deliver a dare, vile dog!
Madam, in Eden I'm Adam
Oozy rat in a sanitary zoo
Ah, Satan sees Natasha
Lisa Bonet ate no basil
Do geese see God?
God saw I was dog
Dennis sinned

12. Type II restriction enzyme nomenclature
• EcoRI – Escherichia coli strain R, 1st
enzyme
• BamHI – Bacillus amyloliquefaciens strain H, 1st
enzyme
• DpnI – Diplococcus pneumoniae, 1st
enzyme
• HindIII – Haemophilus influenzae, strain D, 3rd
enzyme
• BglII – Bacillus globigii, 2nd
enzyme
• PstI – Providencia stuartii 164, 1st
enzyme
• Sau3AI – Staphylococcus aureus strain 3A, 1st
enzyme
• KpnI – Klebsiella pneumoniae, 1st
enzyme
Why the funny names?

13. Results of Type II Digestion
• Enzymes with staggered cuts → complementary ends
• HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’
3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’
• KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’
3’--CCATGG-- 5’ 3’ –C CATGG-- 5’

14. Results of Type II Digestion
• Enzymes that cut at same position on both strands
leave “blunt” ends
• SmaI
5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’
3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’

15. Restriction Endonucleases Cleave DNA
at specific DNA sequences

16. DNA Ligase joins DNA fragments together
• Enzymes that cut with staggered cuts result in
complementary ends that can be ligated together.
• HindIII - leaves 5’ overhangs (“sticky”)
5’ --A AGCTT--3’ 5’ --AAGCTT-- 3’
3’ --TTCGA A--5’ 3’ --TTCGAA-- 5’
• Sticky ends that are complementary (from digests with
the same or different enzymes) can be ligated together.
• Sticky ends that are not complementary cannot be
ligated together.

17. DNA fragments with blunt ends generated by different
enzymes can be ligated together (with lower efficiency),
but usually cannot be re-cut by either original restriction
enzyme.
• SmaI -CCC GGG-
• DraI -AAA TTT-
• Ligations that re-constitute a SmaI or DraI site
(CCCGGG or AAATTT) can be re-cut by SmaI or DraI.
• Mixed ligation products (CCCTTT + AAAGGG) cannot be
re-cut by SmaI or DraI.
DNA Ligase can also join blunt ends
-CCCGGG-
-AAATTT-
-CCCTTT-
-AAAGGG-

18. Any Complementary Ends Can be Ligated
• BamHI -G GATCC-
-CCTAG G-
• BglII -A GATCT-
-TCTAG A-
• Result -GGATCT-
-CCTAGA-
No longer
palindromic, so not
cut by BamHI or BglII

19. Plasmids – vehicles for cloning
• Plasmids are naturally occurring
extrachromosomal DNA molecules.
• Plasmids are circular, double-
stranded DNA.
• Plasmids are the means by which
antibiotic resistance is often
transferred from one bacteria to
another.
• Plasmids can be cleaved by restriction
enzymes, leaving sticky or blunt ends.
• Artificial plasmids can be constructed
by linking new DNA fragments to the
sticky ends of plasmid.
Tetr
Ampr
Ori
pBR322
4361bp
Ori
pUC18
Ampr
MCS
LacZ

20. Cloning Vectors
• A cloning vector is a plasmid that can
be modified to carry new genes.
• Plasmids useful as cloning vectors
must have:
– An origin of replication.
– A selectable marker (antibiotic
resistance gene, such as ampr
and
tetr
).
– Multiple cloning site (MCS) (site
where insertion of foreign DNA will
not disrupt replication or inactivate
essential markers).
– Easy to purify away from host DNA.
Tetr
Ampr
Ori
pBR322
4361bp
Ori
pUC18
Ampr
MCS
LacZ
Older cloning vector
Newer cloning vector

21. Chimeric Plasmids
• Named for mythological beast
(chimera) with body parts from several
creatures.
• After cleavage of a plasmid with a
restriction enzyme, a foreign DNA
fragment can be inserted.
• Ends of the plasmid/fragment are
closed to form a "recombinant
plasmid”.
• Plasmid can replicate when placed in a
suitable bacterial host. Ori
pUC18-hCFTR
Ampr
MCS
LacZ
CFTR

22. DNA cloning requires restriction
endonuclease and DNA ligase
Consider a plasmid with a unique EcoRI site:
5' NNNNGAATTCNNNN 3'
3’ NNNNCTTAAGNNNN 5'
An EcoRI restriction fragment of foreign DNA
can be inserted into a plasmid having an EcoRI
cloning site by:
a) cutting the plasmid at this site with EcoRI,
b) annealing the linearized plasmid with the
EcoRI foreign DNA fragment, and,
c) sealing the nicks with DNA ligase.
5' NNNNGAATTCNNNN 3'
3' NNNNCTTAAGNNNN 5’
This results in a recombinant DNA molecule.

23. Introduction to Cloning and Recombinant
Technology: Lecture Outline
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics

24. Key features of DNA replication
are used in DNA sequencing
• DNA synthesis occurs in the 5´ to 3´ direction.
• DNA synthesis requires a template and a primer.
• DNA replication is semi-conservative (one strand copied).
• DNA replication is carried out by an enzyme called DNA
polymerase.

25. DNA synthesis requires a 3´-OH to make the next
phosphodiester bond during DNA synthesis
normal
dNTP

26. Dideoxy NTPs block DNA synthesis
H

27. H H
A nucleotide-
specific stop in
DNA synthesis
A 3´-OH on the last ribose is needed for DNA synthesis
ddNTPs block formation of the next
phosphodiester bond during DNA synthesis
ddNTP

28. A mixture of dNTPs and ddNTPs are
used in DNA sequencing

29. Polyacrylamide gel electrophoresis is used to
visualize the results of the sequencing reaction

30. Automated DNA sequencing with
fluorescent dyes coupled to each reaction
Fluorescent dye coupled to
reaction allows visualization
of di-deoxy termination
events by means of a laser
that detects the colored
product.
This shows four different
reactions as done with the
old manual sequencing.

31. Automated DNA sequencing output-
4 reactions carried out in one tube

32. Molecular Medicine: The Human Genome Project
NCBI.nlm.nih.gov/genome/guide/human/index
3.2x109
nucleotide pairs

33. Technology now exists to sequence everyone’s DNA
Took just 4 months,
$1.5 million to obtain
the entire DNA
sequence of James
Watson.

34. The genomes of many organisms have been sequenced

35. Genome resources for many
organisms are available

36. Introduction to Cloning and Recombinant
Technology: Lecture Outline
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics

37. Understanding the arrangement of genes
may help understand disease

38. Southern blot: One way to detect genome
structure and disease markers in genomic DNA
-Purify genomic DNA
-Digest with restriction enzyme
-Run agarose gel

39. Restriction fragment length polymorphisms
(RFLPs) can be associated with disease alleles
70% of carriers of the sickle cell
gene have a 13.0 kb HpaI fragment.
30% of carriers have 7.0 kb HpaI
fragment
HpaI Digest
Nor-
mal
Variants
1 2 3
Consider two alleles of a gene.
Allele A has 3 BamHI sites, while
allele a has only two BamHI sites.
Southern Blot
probe

40. Direct Detection of a Sickle Cell
Mutation by RFLP
Wild Type Mutant
Pro Glu Pro Val
CCT GAG CCT GTG
DdeI site no DdeI site
Gene encoding Wild type β-subunit
Gene encoding sickle cell β-subunit
AS AS SS AA
A specific hemoglobin mutation
[DdeI cuts at CTNAG]

41. • Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Introduction to Cloning and Recombinant
Technology: Lecture Outline

42. Polymerase Chain Reaction (PCR)
• Allows quick identification of genetic markers:
Identify bacteria in infections
Identify viruses in virus infections
Paternity testing, genetic counseling, forensics
 Can exclude individuals, but cannot prove guilt.
• Requires only small amounts of DNA.
• A repetitive DNA synthesis reaction.
• Thermostable DNA polymerase:
Isolated from bacteria in hot springs or near thermal vents
in the deep ocean.
• Requires gene-specific DNA primers and
deoxyribonucleotide triphosphates (dNTPs).

43. A thermophilic (heat-loving) bacteria called Thermus aquaticus is
the source of Taq DNA polymerase used in PCR reactions.
Polymerase Chain Reaction (PCR)

44. The first round of PCR
94°C 37-65°C 70-75°C

45. PCR increases the yield of DNA exponentially

46.  Begins with DNA containing a sequence
to be amplified and a pair of synthetic
oligonucleotide primers that flank the
sequence.
 Next, denature the DNA to single strands
at 94˚C.
 Rapidly cool the DNA (37-65˚C) and
anneal primers to complementary single
strand sequences flanking the target DNA.
 Extend primers at 70-75˚C using a heat-
resistant DNA polymerase such as Taq
polymerase derived from Thermus
aquaticus.
 Repeat the cycle of denaturing, annealing,
and extension 20-45 times to produce 1
million (220
) to 35 trillion copies (245
) of the
target DNA.
 Extend the primers at 70-75˚C once more
to allow incomplete extension products in
the reaction mixture to extend completely.
 Cool to 4˚C and store or use amplified
PCR product for analysis.
A typical PCR protocol

47. PCR cycle 28 – ~1 billion strands
5´3´
B
3´ 5’
5´ 3´
5’ 3´
A
3´ 5´
BA
3´5´
BA
2 original strands.
28 strands starting with primer A, but with no
end.
28 strands starting with primer B, but with no
end.
~500,000,000 strands starting with primers A
(5´) and ending with primer B (referred to as
unit-length strand in previous figure).
~500,000,000 strands starting with primer B
(5´) and ending with primer A (referred to as
unit-length strand in previous figure).

48. PCR in Medicine
• Since 1987, PCR has had a major impact on prenatal
diagnosis of single gene disorders.
• Also very important in carrier testing for genetic
diseases.
• Improved speed, accuracy and technical flexibility over
previous methods.

49. PCR and prenatal diagnosis
• For prenatal diagnosis, PCR used to amplify DNA from fetal cells
obtained from amniotic fluid.
• Single base changes then detected by one or more of following:
-dot blot (spot hybridization) with oligonucleotides specific for
known mutation.
-restriction enzyme analysis (RFLP).
-direct sequencing of DNA.
• Important to be certain of result so combination of two methods
provides confirmation.
• Many other conditions can be detected with same approach,
including:
-Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia,
Huntingdon's disease, Duchenne muscular dystrophy (DMD).

50. PCR to detect HIV
• PCR allows the direct detection of HIV genomes in patient
blood before the appearance of HIV antibodies.
• viral DNA/RNA only represents a minute proportion of total cell
DNA.
• Only a small fraction of blood cells are infected (1/10,000).
• also require high degree of specificity while targeting conserved
regions of DNA to guard against high level of genetic variability
characteristic of retroviruses.
• High risk of cross-contaminating sample with small amounts of
amplified DNA from previous sample requires extra precautions
to prevent false-positives.
• PCR can detect 10-20 copies of viral DNA from 150,000 human
cells.

51. PCR can be more rapid and accurate
than other diagnostic tests
• Diagnosis of the middle ear infection known as otitis media. The
technique has detected bacterial DNA in children's middle ear fluid,
signaling an active infection even when culture methods failed to
detect it.
• Lyme disease, the painful joint inflammation caused by bacteria
transmitted by tick bites, can be diagnosed by detecting the disease
organism's DNA contained in joint fluid.
• PCR is the most sensitive and specific test for Helicobacter pylori,
the disease organism now known to cause almost all stomach
ulcers.
• PCR can detect three different sexually transmitted disease
organisms on a single swab (herpes, papillomaviruses, and
chlamydia).

52. PCR in Forensics
Crucial forensic evidence may be present in very small quantities.
• often too little material for direct DNA analysis.
• but PCR can generate sufficient DNA from a single cell.
• PCR also possible on extensively degraded DNA.
• examples include DNA from single dried blood spot, saliva (on cigarette
butt), semen, tissue from under fingernails, hair roots.
Other advantages of PCR in forensic science are:
• relatively simple to perform and simple to standardize.
• results obtainable within 24 hours.
The major legal problems with PCR are the potential for cross-
contamination between samples and the complexity of explaining what
the results mean to the jury.

53. PCR can exclude suspects
but cannot prove guilt
• DNA typing is only one of many pieces of evidence that can lead to a
criminal conviction, but it has proved invaluable in demonstrating innocence.
• Sometimes seemingly strong DNA evidence does not lead to a conviction
(see O.J. Simpson trial).
• Dozens of cases have involved people who have spent years in jail for
crimes they did not commit until PCR exonerated them.
• Even when evidence such as semen and blood stains are years old, PCR
can make unlimited copies of the tiny amounts of DNA remaining in the
stains for typing.

54. Variable Number of Tandem Repeat (VNTR)
analysis is commonly used in forensics
VNTR is based on hypervariable microsatellite sequence polymorphisms
within the human genome. These sequences (e.g., CACACA …) are found
in many locations in the human genome and vary greatly from person to
person.

55. Using VNTR to compare forensic and
suspect samples
Individuals A & C are
excluded by this
analysis. The samples
from individual B will be
subjected to further tests.

56. Conclusions
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Questions?