Section J

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Section J Analysis and uses of cloned DNA
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Plant cell
Animal cell
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Contents

• J1 Characterization of clones
• Characterization, Restriction mapping,
  Partial digestion, Labeling nucleic acid,
  Southern and Northern blotting
• J2 Nucleic acid sequencing
• DNA sequencing, RNA sequencing, Sequence
  databases, Analysis of sequences, Genome
  sequencing projects
• J3 Polymerase of cloned genes
• PCR, the PCR cycle, Template, Primers,
  Enzymes, PCR optimization, PCR variations
• J4 Organization of cloned genes
• Organization, Mapping cDNA on genomic DNA, S1
  nuclease mapping, Primer extension, Gel
  retardation, DNase? footprinting, Reporter genes
• J5 Mutagenesis of cloned genes
• Deletion mutagenesis, Site-directed
  mutagenesis, PCR mutagenesis
• J6 Applications of cloned genes
• Applications, Recombinant protein,
  Genetically modified organisms, DNA
  fingerprinting, Medical diagnosis, Gene therapy

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J1 Characterization of clones
Characterization
Preparation of pure DNA is the first step of
any characterization. Plasmid DNA from bacterial
colonies Bacteiophage DNA Plaque purified
phage ? ? infecting a bacterial culture ? ? cell
lysis ? ? phage particles ? ? phenol-chloroform,
ethanol precipitate ? ? Bacteiophage DNA
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J1 Characterization of clones
Restriction mapping
Example1
Digests Resultant Fragments
EcoRI 3 kb, 5 kb
HindIII 2 kb, 6 kb
EcoRI HindIII 2 kb, 1 kb, 5 kb

• The most common application of restriction
  mapping is presented Determining the orientation
  of a cloned insert. This method requires that
  restriction maps of the cloning vector and the
  insert are already available.

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J1 Characterization of clones
Restriction mapping
Example2
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J1 Characterization of clones
Partial digestion
2kb
4kb
3kb
1kb
10 kb insert
Complete digestion
Partial digestion
10 kb
7 kb
6 kb
4 kb
Can not delineate the restriction sites.
3 kb
2 kb
1 kb
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Delineate the restriction sites by partial
digested end-labeled radioactive DNA.
E
E
E
3kb 1kb 2kb
4kb

10 kb insert

6 kb

4 kb
3 kb

10 kb
End-labeled radioactive DNA
6 kb
4 kb
Partial digestion
3 kb
Agarose electrophoresis
Autoradiography
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J1 Characterization of clones
Labeling nucleic acid
Radioactive labeling display and/or magnify the
signals by radioactivity Non-radioactive
labeling display and/or magnify the signals by
Biotin and digoxin etc
1.End labeling put the labels at the
ends 2.Uniform labeling put the labels
internally
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• End labeling
• (1)Single stranded DNA/RNA

5-end labeling dephosphorylation ?
polynucleotide kinase 3-end labeling
terminal transferase
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(2)Double stranded DNA/RNA
Fill in the recessive 3-ends( 3-??) by DNA
polymerase. Labeled at both ends
---------------------G ---------------------CTTAAp
5
5pAATTC G
For restriction mapping, cut the DNA with
another enzyme
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2. Uniformly labeling of DNA/RNA
(1)Nick translation (????) DNase I to
introduce random nicks ?DNA Pol I to remove dNTPs
from 5 to 3 and add new dNTP including labeled
nucleotide at the 3 ends.
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(2)Hexanucleotide primed labeling(?????????,random
labeling ????) Denature DNA ? add random
hexanucleotide primers and DNA pol ? synthesis of
new strand incorporating labeled nucleotide.
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3. Specific probes
(1)Strand-specific DNA probes e.g.M13 DNA
as template the missing strand can be re-
synthesized by incorporating radioactive
nucleotides.
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(2)Strand-specific RNA probes
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J1 Characterization of clones
Southern and Northern blotting

1. Southern blotting, for detecting DNA
2. Northern blotting, for detecting RNA
3. Western blotting, for detecting protein.

Blot type Target Probe Applications
Southern DNA DNA or RNA mapping genomic clonesestimating gene numbers
Northern RNA DNA or RNA RNA sizes, abundance,and expression
Western Protein Antibodies protein size, abundance
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1.Genomic DNA preparation 2.Restriction
digestion 3.Denature with alkali 4.Agarose gel
electrophoresis 5.DNA blotting/ transfer and
fixation 6.Probe labeling 7.Hybridization
(temperature) 8.Signal detection (X-ray film or
antibody)
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Southern analysis
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Northern blotting
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J2 Nucleic acid sequencing
DNA sequencing

• Three main methods
• 1. Maxam and Gilbert chemical method
• ? 2. Sangers enzymatic method
• ? 3. Sequencing by hybridization (SBH)

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1. Maxam and Gilbert chemical method

• The end-labeled DNA is subjected to
  base-specific cleavage reactions prior to gel
  separation.
• Modification of bases
• Methylation by dimethyl sulfate G (DMS)
• Formic acid Purines A G
• Hydrazine hydrolyze T C
• Hydrazine high salt only C

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A A A G A T T A A G C C
Dimethyl sulfate Formic acid Hydrazine
Hydrazinehigh salt
G AG CT C
?????
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2. Sangers enzymatic method
Uses dideoxynucleotides as chain terminators to
produce a ladder of molecules generated by
polymerase extension of primer
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C
G
T
A
Sangers method Template primer
(15-17nt) dNTPs ddNTPs 35SdATP T7 DNA
pol PAGE Autoradiography
3GTGACTACTCAGGCACTTGCTTTGCC5
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Automatic sequencer
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3. Sequencing by hybridization(SBH)
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J2 Nucleic acid sequencing
RNA sequencing

• By base-specific cleavage of 5-end-labeled RNA
  using RNases that cleave 3 to a particular
  nucleotide. Partial digestion is required to
  generate a ladder of cleavage products which are
  analyzed by PAGE.

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RNase T1 cleaves after G RNase U2 after
A RNase Phy M after A and U Bacillus cereus
RNase after U and C
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J2 Nucleic acid sequencing
Sequence databases

• DDBJ(??????????) http//www.ddbj.nig.ac.jp
• EMBL-EBI (?????????) http//www.ebi.ac.uk/Databa
  ses/index.html
• Genbank at NCBI (????????????)
• http//www.ncbi.nlm.nih.gov

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J2 Nucleic acid sequencing
Analysis of sequences

• Using computers and software packages, such as
  GCG sequence analysis package.
• 1. Identify important sequence features such as
  restriction sites, open reading frames, start and
  stop codons, as well as potential promoter sites,
  intron-exon junctions, etc.

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ORF 2
ORF 1
100
200
300
400
500
600
700
Sequence analysis of a cloned DNA sequence
revealed some important features
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2. Homology search by BLAST (NCBI) or FASTA
(EBI) Compare new sequence with all other
known sequences in the databases, which can
determine whether related sequences have been
obtained before.
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J2 Nucleic acid sequencing Genome
sequencing projects

• With the development of automated DNA sequencers
  and robotic workstations to prepare samples for
  sequencing, the entire genome sequence of several
  organisms have been determined.
• Many phages and viruses
• Several Bacteria (E. coli, 4 x 106)
• Plant (Arabidopsis 6.4 x 107 , rice)
• Human 3.3 x 109

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J3 Polymerase of cloned genes PCR

• The polymerase chain reaction (PCR)
• To amplify a sequence of DNA using a pair of
  primers each complementary to one end of the DNA
  target sequence.

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J3 Polymerase of cloned genes
the PCR cycle

• Denaturation The target DNA (template) is
  separated into two stands by heating to 95?
• Primer annealing The temperature is reduced to
  around 55? to allow the primers to anneal.
• Polymerization (elongation, extension) The
  temperature is increased to 72? for optimal
  polymerization step which uses up dNTPs and
  required Mg.

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J3 Polymerase of cloned genes Template

• Single-or double-stranded form
• The size of the template DNA is not critical
• In the case of mammalian or plant genomic DNA, up
  to 1.0 ug of DNA is utilized per reaction. The
  typical amounts of yeast, bacterial, and plasmid
  DNAs used per reaction are 10 ng, 1ng, and 1pg,
  respectively
• Template DNA is dissoved in 10 mM Tris-Cl (pH
  7.6) containing a low concentration of EDTA (lt0.1
  mM).

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J3 Polymerase of cloned genes Primers

• PCR primersabout 18 to 30 nt long and with
  similar GC contents.
• Tm2(at)4(gc) determine annealing
  temperature. If the primer is 18-30 nt, annealing
  temperature can be Tm-5oC.

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If the target DNA is not known,there is only
limited amino acid sequence available. Degenerate
primers An oligo pool derived from protein
sequence. E.g. His-Phe-Pro-Phe-Met-Lys can
generate a primer CAU(CAC)-UUU(UUC)-CCU(CCC,CCA,C
CG)- UUU(UUC)-AUG-AAA(AAG) 2x2x4x2x2 64
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J3 Polymerase of cloned genes Enzymes

• The most common is Taq polymerase from Thermus
  aquaticus. It has no 3 to 5 proofreading
  exonuclease activity. Accuracy is low, not good
  for cloning.
• Pfu (Pyrococcus furiosus, Promega Stratagene),

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J3 Polymerase of cloned genes
PCR optimization

• PCR cycle
• Enzymes
• Template DNA
• Mg

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J3 Polymerase of cloned genes
PCR variations

• 1. Inverse PCR, IPCR
• 2. Anchored PCR, APCR
• 3. asym metric PCR
• 4. Reverse transcription RT-PCR
• 5. ????PCR
• 6. Nest PCR
• 7. multiplex PCR
• 8. ??PCR
• 9. differential PCR, d-PCR
• 10. quantitative PCR, qPCR
• 11. in situ PCR
• 12. immuno-PCR
• 13. Thermal Asymmetric Interlaced PCR,TAIL-PCR

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J4 Organization of cloned genes
Organization

• The absent sequences are usually introns and
  sequences upstream of the transcription start
  site and down stream of the 3-processing site.
• Start and stop sites for transcription
• regulatory sequences.

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J4 Organization of cloned genes Mapping
cDNA on genomic DNA

• The genomic clone is digested on a gel and then
  subjected to Southern blot using all or part of
  the cDNA as a probe. Show which genomic
  restriction fragments contain cDNA sequences
• Using a probe from one end of a cDNA can show
  the polarity of the gene in the genomic clone.
• Some of the restriction sites will be common in
  both clones but may be different distances apart.

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J4 Organization of cloned genes
S1 nuclease mapping

• Determines the precise 5- and 3- ends of RNA
  transcripts. Sequence ladder is required to
  determine the precise position.

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J4 Organization of cloned genes
Primer extension

• A primer is extended by a polymerase until the
  end of the template is reached and the polymerase
  dissociated.
• The length of the extended product indicates the
  5end of temple.

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J4 Organization of cloned genes
Gel retardation

• Mixing a protein extract with a labeled DNA
  fragment and running the mixture on a native gel
  will show the presence of DNA-protein complex as
  retarded bands on the gel.

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J4 Organization of cloned genes
DNase? footprinting

• The footprint of a protein bound specifically
  to a DNA sequence can be visualized by treating
  the mixture of end-labeled DNA plus protein with
  small amounts of DNase I prior to running the
  mixture on a gel.
• The footprint is a region with few bands in a
  ladder of cleavage products.

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J4 Organization of cloned genes
Reporter genes

• To study the function of a control element of a
  gene like HSP70 (promoter and regulatory
  elements). Reporter genes such as ß
  -galactosidase or luciferase to report the
  promoter action.

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J5 Mutagenesis of cloned genes
Deletion mutagenesis

• In the cDNA clones,it is common to delete
  progressively from the ends of the coding region
  to discover which parts of the whole protein have
  particular properties.

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Exunuclease III Unidirectional deletion using
exonuclease III.
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J5 Mutagenesis of cloned genes
Site-directed mutagenesis

• Formerly, single-stranded templates prepared
  using M13 were usedPrimer oligonucleotide with
  desired mutation, extension by DNA polymerase,
  then ligation.
• Now PCR techniques are now preferred

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J5 Mutagenesis of cloned genes
PCR mutagenesis

• By making forward and reverse mutagenic primers
  and using other primers that anneal to common
  vector sequence, two PCR reactions are carried
  out to amplify 5- and 3- portions of the DNA to
  be mutated.
• The tow PCR products are mixed and used for
  another PCR using the outer primers only-Part of
  this product is then subcloned to replace the
  region to be mutated in the starting molecule.

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J6 Applications of cloned genes Applications

• Recombinant protein production
• Genetically modified organisms
• DNA fingerprinting
• Diagnostic kits
• Gene therapy

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J6 Applications of cloned genes
Recombinant protein

• Recombinant proteins Growth hormone, insulin for
  diabetes,interferon in some immune
  disorders,blood clotting factor VIII in for
  hemophilia.

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J6 Applications of cloned genes
Genetically modified organisms

• Introducing a foreign gene into an organism which
  can propagate creates a genetically modified
  organism.
• Transgenic sheep have been crested ro produce
  foreign proteins in their milk.
• Cloned genes are introduced into germ cells.

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J6 Applications of cloned genes
DNA fingerprinting

• Hybridizing southern blots of genomic DNA with
  probes that recognize simple nucleotide repeats
  gives a pattern that is unique to an individual
  and can be used an a fingerprint.
• This has applications in forensic science, animal
  and plant breeding and evolutionary studies.
• Simple nucleotide repeats vary in number between
  individuals but are inherited.

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J6 Applications of cloned genes
Medical diagnosis

• The sequence information derived from cloning
  medically important genes has allowed the design
  of many diagnostic test kit which can help
  predict and confirm a wide range of disorders.
• By using sequence information to screen patients.
  

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J6 Applications of cloned genes
Gene therapy

• Attempts to correct a genetic disorder by
  delivering a gene to a patient are described as
  gene therapy.
• To treat some genetic disorders by delivering a
  normal copy of the defective gene to patients.
  The gene can be cloned into a virus that can
  replicate but not cause infection.

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Multiple choice questions

• 1. A linear DNA fragment is (100) labeled at
  one end and has 3 restriction sites for EcoRI. If
  it is partially digested by EcoRI so that all
  possible fragments are produced how many of these
  fragments will be labeled and how many will not
  be labeled?
• A 4 labeled 6 unlabeled.
• B 4 labeled 4 unlabeled.
• C 3 labeled 5 unlabeled.
• D 3 labeled 3 unlabeled.
• 2. Which of the following are valid methods of
  labeling duplex DNA?
• A 5'-end labeling with polynucleotide kinase.
• B 3'-end labeling with polynucleotide kinase.
• C 3'-end labeling with terminal transferase.
• D 5'-end labeling with terminal transferase.
• E nick translation.

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• 3. Which one of the following statements about
  nucleic acid sequencing is correct?
• A. the Sanger method of DNA sequencing involves
  base specific cleavages using piperidine.
• B. the Maxam and Gilbert method of DNA
  sequencing uses a DNA polymerase and chain
  terminating dideoxynucleotides.
• C. enzymatic sequencing of RNA uses RNases A,
  T1, Phy M and B. cereus RNase.
• D enzymatic sequencing of DNA uses a primer
  which is extended by an RNA polymerase.
• E enzymatic sequencing of RNA uses RNases T1,
  U2, Phy M and B. cereus RNase.
• 4 Which one of the following statements about
  peR is false?
• A the PCR cycle involves denaturation of the
  template,annealing of the primers and
  polymerization of nucleotides.
• B PCR uses thermostable DNA polymerases.
• C ideally PCR primers should be of similar
  length and GC content.
• D PCR optimization usually includes varying the
  magnesium concentration and the polymerization
  temperature.
• E if PCR was 100 efficient, one target molecule
  would amplify to 2n after n cycles.

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• 5. Which two of the following statements about
  gene mapping techniques are true?
• A. S1 nuclease mapping determines the
  nontranscribed regions of a gene.
• B. primer extension determines the 3'-end of a
  transcript.
• C. gel retardation can show whether proteins can
  bind to and retard the migration of a DNA
  fragment through an agarose gel.
• D. DNase I footprinting determines where on a
  DNA fragment a protein binds.
• E the function of DNA sequences in the promoter
  of a gene can be determined if they are ligated
  downstream of a reporter gene and then assayed
  for expression.
• 6. Which one of these statements about
  mutagenesis techniques is false?
• A. exonuclease III removes one strand of DNA in
  a 5' to 3' direction from a recessed 5'-end.
• B. exonuclease III removes one strand of DNA in
  a 3' to 5' direction from a recessed 3'-end.
• C. mutagenic primers can be used in PCR to
  introduce base changes.
• D. mutagenic primers can be used with a single
  stranded template and DNA polymerase to
  introduce base changes.
• E. deletion mutants can be created using
  restriction enzymes.

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• 7. Which one of these statements about the
  applications of gene cloning is false?
• A large amounts of recombinant protein can be
  produced by gene cloning.
• B DNA fingerprinting is used to detect proteins
  bound to DNA.
• C cloned genes can be used to detect carriers of
  disease-causing genes.
• D gene therapy attempts to correct a disorder by
  delivering a good copy of a gene to a patient.
• E genetically modified organisms have been used
  to produce clinically important proteins.

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