Objectives
? Recombinant DNA
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? Probes
? Restriction map
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? Gene cloning? Gene library
? Cloning vectors
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? RFLP
? DNA Fingerprinting
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? DNA foot printing? Genomic imprinting
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Three techniquesthat facilitate
analysis of human DNA
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Steps of Genetic Engineering
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? Cutting DNA at a specific site? Joining of two DNA fragments to create a
novel DNA
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? Cloning or amplification of available DNA? Expression of a DNA to obtain its product
? Sequencing of a DNA molecule
? Synthesis of an oligonucleotide
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4Application of Recombinant
Technology
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? Understanding of diseases: Sickle cellanaemia, Thalassemia
? Diagnosis of diseases: AIDS, Hepatitis
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? Treatment of Diseases: Human Insulin? Prevention of diseases: Hepatitis vaccines
? Gene therapy: SCID
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Recombinant DNA
Two DNA fragments of interest: Even from different source or species
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Cohesive or sticky ends with complementary sequencesTreated with same RE
in some cases blunt ends are joined by
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Homopolymer tailing
A small synthetic duplex oligonucleotide having RE sites attached
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5` ends of the linker DNA are phosphorylated by polynucleotide kinase6
Different Enzymes used in DNA Recombinant
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technology EnzymeFunctions
DNA ligase
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joins of ends
DNA Pol I
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Synthesis of doubleRestriction
stranded DNA
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Endonuclease
DNAse I
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Produces nicks insDNA
Exonuclease I I
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Removes nt from 3`
end
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exonucleaseRemoves from 5` end
Polynucleotide kinase Phosphorylates 5` OH
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group
Alk phosphatase
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Removes 5` PO4S1 nuclease
Degrades sDNA
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Recognition sequence of
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restriction endonucleaseEcoRI shows
two-fold rotational symmetry
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Nomenclature of
Restriction endonuclease
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Why they are named so
What are sticky ends and
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blunt endsRestriction sites frequency
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TaqI and Hae I I I
(Haemophilus aegyptius)
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restriction endonucleases10
Restriction Map
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involves the size analysis of restriction fragmentsproduced by several restriction enzymes
individually and in combination
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Probe
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15-20 nt long oligont used to search a particular DNA fragment
Chemically synthesized DNA or RNA pieces
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Generally labelled with radioactive material or a fluoroscent labelConstruction of a probe
From Genetic database
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homologous gene in other species : heterologous gene probe
mRNA
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protein----- rich in Trp and Met13
Label ing of a Probe
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End labelling: 32P
Random labelling: During synthesis: Usually GTP:
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Fluorescent labelled14
End-labelling of a gene probe at the 5' end with
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alkaline phosphatase and polynucleotide kinase15
End-labelling of a gene probe at the 3'
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end using terminal transferase
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Cloning
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Process of producing large number of identical copies from
one single original DNA molecule or fragment
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ImportanceTo study gene: purified form and in sufficient amount
To study sequencing, expression in different tissues
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under different conditions
Methods of amplification
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Cell basedCell free
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Outline of gene cloning
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Gene Library
? All of the DNA extracted from an organism
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and digested with a restriction enzyme willresult in a collection of clones. This collection
of clones is known as a gene library
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Type of gene library
Genomic library: Total chromosomal DNA of organism
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cDNA library: represents the mRNA from a cell or tissue
at a specific point in time
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Type of gene library depends on final application of DNAGoal: Production of new or modified proteins or determination
of tissue specific expression and timing pattern
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cDNA library
Goal: To understand the control of protein production for a
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particular geneGenomic library
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Gene Library
? 1. Construction of gene library
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? Digesting genomic DNA molecules? Choice of Enzyme?
? Ligating DNA molecules
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? Carried out at 10o C to lower the kinetic energy
and to reduce the chances of sticky ends
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parting? 2. Cloning vectors
? 3. Screening Gene Library
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Numbers of clones required for representation of DNA
in a genome library
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Ligation molecules with cohesive ends. Complementary cohesive ends base -pair,
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forming a temporary link between two DNA fragments. This association of fragmentsis stabilised by the formation of 3' to 5' phosphodiester linkages between cohesive ends,
a reaction catalysed by DNA ligase.
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Comparison of general steps in the construction of
genomic and cDNA library
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Cloning Vector
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. DNA elements that may be stably maintainedand propagated in a host organism for which
the vector has replication functions.
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. Host organism is a bacterium such as E. coli
. vector with a replication origin in E. coli will
replicate (together with any incorporated
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DNA) efficiently26
Comparison of vectors general y available for cloning
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VectorHost cell
Vector structure Insert
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range(kb)
M13
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E coliCircular virus
1-4
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Plasmid
E coli
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Circular plasmid1-5
Phage
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E coli
Linear virus
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2-25Cosmids
E coli
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Circular plasmid
35-45
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BACsE coli
Circular plasmid
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50-300
YACs
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S.Linear
100-2000
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Cerevisiae chromosome
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Structure of E.Coli plasmid cloning vector pBR32228
Replica plating to detect recombinant
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plasmids29
Map and important features of pUC18
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Principle of blue/white selection for
the detection of recombinant vectors.
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Cosmid vectors
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Cosmid vectors incorporate the cos sites from phage l and alsothe essential features of a plasmid, such as the plasmid origin
of replication, a gene for drug resistance, and several unique
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restriction sites for insertion of the DNA to be cloned.
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Expression vector
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The inserted sequence must be placed so that
its reading frame is in phase with the regulatory sequence
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33Shuttle vectors
? Shuttle vectors have origins of replication for
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yeast and bacteria such as E. coli. This means
that constructs may be prepared rapidly in the
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bacteria and delivered into yeast forexpression studies.
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Delivery of vectors into Eukaryotes? Transfection:
? to deliver recombinant molecules into animal cel s
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? 1. making the membrane permeable with
divalent cations / use of polyethylene glycol (PEG)
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? 2. electroporation: subjected to pulses of a high-voltage gradient
? 3. lipofection: DNA is encapsulated by a core of
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lipid-coated particles
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SCREENING GENE LIBRARIES36
Colony hybridisation technique
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for locating specific bacterialcolonies harbouring recombinant
plasmid vectors containing
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desired DNA fragments.
Application of gene cloning
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Molecular analysis of diseaseNormal gene variation--Polymorphism
Gene variation causing disease--Beta globin gene
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Detection of point mutation---Sickle cell disease
Detection of deletion /insertion/rearrangement--
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Beta globin genePrenatal diagnosis
Preimplantation diagnosis: done in IVF
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Disease linkage analysis--Microsatel ite repeats in families
Forensic medicine
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38Structural Alterations of the a-Globin Gene
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Schematic representation of the -globin gene cluster andof the lesions in some genetic disorders.
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Sickle cel disease41
Pedigree analysis of Sickle cel disease
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Restriction Fragment Length Polymorphism and SNPs
major use of SNPs/RFLPs is in the definition of inherited diseases
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in which the functional deficit is unknown
SNPs/RFLPs can be used to establish linkage groups, which in turn,
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by the process of chromosome walking,will eventually define the disease locus
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The technique of chromosome walking
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Microsatellite repeat variation in some diseases
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Disease
Repeat
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Normal lengthMutation length
of repeats
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Fragile X
(CGG)n
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6-54200-1000
syndrome
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Fredriech ataxia (GAA)n
7-22
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7200Myotonic
(CTG)n
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50-35
50-4000
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dystrophySpino cerebel ar (CAG)n
19-36
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43-81
ataxia
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45? DNA Finger printing
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? Alec Jeffrey in 1984? Each individual has unique sequences
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51Purpose
? 1. Paternity dispute
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? 2. Criminal identification52
Method
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? Isolation of DNA? Digestion of DNA by RE
? Amplification
? Separation by gel electrophoresis
? Blotting
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? Hybridisation with radiolabelled probe? Autoradiography
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5455
? DNA Footprinting
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56Footprinting with DNase I
? Footprinting enables a control region to be
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positioned within a restriction fragment that hasbeen identified by gel retardation.
? Footprinting works on the basis that the
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interaction with a regulatory protein protects the
DNA in the region of a control sequence from the
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degradative action of an endonuclease such asdeoxyribonuclease (DNase) I.
? This phenomenon can be used to locate the
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protein binding site on the DNA molecule.
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A bound protein can protect aregion of DNA that is much longer
than the control sequence.
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Genomic imprinting
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What Mendel found out
? Two parents make equal contribution to the
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character
? The effect of an allele is independent of
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whether it comes from the female or malegamete
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Summary
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? Recombinent DNA is the joining of two fragments cut with same restrictionendonuclease
? Probe is a 15-20 nt long oligonucleotide used to search a particular DNA fragment
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? Cloning is a process of producing large number of identical copies from one single
original DNA molecule or fragment
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? Type of gene library depends on final application of DNA? Delivery of vector in bacteria is cal ed transformation and in animal cel s is
transfection
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? RFLP is a technique used to identify the individual as wel as to detect disease
condition such as Sickle cel anaemia
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? DNA finger printing is to identify the individual based on differential tandemrepeat sequences
? Footprinting works on the basis that the interaction with a regulatory protein
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protects the DNA from the degradative action of an endonuclease such as
deoxyribonuclease (DNase) I.
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? Genomic imprinting usual y uses DNA methylation (epigenetic regulati on).70