Recombinant DNA technology
Restriction enzymes are used for cutting DNA at
precise locations
Vectors are used for the propagation of cloned DNA
fragments
Libraries of cloned DNA fragments allow searching for a
specific DNA fragment
Restriction Enzymes
Nucleases:
Exonuclease,digests from ends
Endonuclease,cleaves internally
Restriction Enzyme,They are synthesized by bacteria
to protect themselves from invading viruses.
class of endonucleases which cleaves DNA molecules at
specific nucleotide sequences called restriction sites,
1978 Nobel Prize,Arber,Smith & Nathans
Naming of enzymes
Over 150 different restriction endonucleases have been
identified.
Isolated from different bacteria
Recognize specific DNA sequences which are usually
4-8 bp long
Escherichia coli yields several different enzymes,one
is called EcoRI,The E is derived from the genus,and
the second and third letters (co) # from the species
name,The numbers refer to the strain,in this case
RY13.
Restriction Enzyme Specificity
e.g,Eco RI,GAATTC
Bam HI GGATCC,6-cutters”
Frequency,46 = average of 1 cut site per 4,096 bp
e.g,Sau 3A GATC,4-cutter”
Frequency,44 = average of 1 cut site per 256 bp
e.g,Not I GCGGCCGC,8-cutter”
Frequency,48 = average of 1 cut site per 65,536
Palindromes
Madam I’m Adam
madA m’I madaM
Blunt ends and sticky ends
,Blunt ends”
e.g,Sma I,Hae III,any other blunt-cutter
CCCGGG
GGGCCC
GGCC
CCGG
CCC
GGG
CC
GG
+ CCCCCGGGGG
Plasmid,Cloning Vectors
Origin of replication
Selectable marker
Cloning sites (unique)
Shuttle vectors
grow in more than one host
cell
Construction of Recombinant DNA Libraries
Genomic library:
A collection of clones representing the entire
chromosomal DNA sequence,These clones include
the entire gene,including introns (eukaryotes) and
regulatory regions.
cDNA library (complimentary DNA)
A collection of clones representing DNA copies of all
the different mRNA molecules in the cell,These
clones do not include the introns (eukaryotes) nor the
regulatory regions of the gene because they are
derived from mRNA.
Genomic Library Construction
Overlapping clones:
Random shearing
Partial digests,not all sites are cut
use 4-bp recognition (e.g.,Sau 3A) or rare-cutting enzymes
(notI; 8 bp recognition)
S S S S S S S S S S S S S S S S S S S S S
Genomic Libraries
Sometimes called clone banks or gene banks
To obtain a complete library the number of individual
clones required in a library is dependent upon:
size of the genome (the larger the more needed)
the size of the DNA insert in the cloning vector
(smaller insert sizes require more individuals)
Example,Human genome (3 billion bp) with an insert
size of 40,000 bps = 330,000 clones,But,if the insert
size is 200,000 bps,only 6,900 clones are needed to
represent the entire human genome,
Screening a library for a specific clone
NEED A PROBE!
Complementary DNA or RNA sequence
Use radioactive DNA to detect the
presence of the clone
fluorescent antibodies can also be used
to look for specific proteins
Isolating
mouse CF
gene with
human CF
probe
Characterization of cloned fragments
Construct a map of the fragment that shows the
location of all restriction endonuclease cut sites.
Determine the area within the clone that codes for a
specific gene.
Determine the sequence of nucleotides in the clone.
Genotyping with molecular biology
techniques
Genotyping with microsatellite
Detecting single base difference or SNP (single
nucleotide polymorphism)
RFLP,restriction fragment length polymorphism,
RFLP can arise from single base pair change and
used for its detection,RFLP can also arise from
other DNA changes (deletion or insertion),
SSCP,single strand conformation polymorphism
ASO,allele specific oligo
RAPD
RAPD,random amplification of polymorphic DNA
Used for isolating single-base-pair polymorphic
markers of unknown genome
Restriction enzymes are used for cutting DNA at
precise locations
Vectors are used for the propagation of cloned DNA
fragments
Libraries of cloned DNA fragments allow searching for a
specific DNA fragment
Restriction Enzymes
Nucleases:
Exonuclease,digests from ends
Endonuclease,cleaves internally
Restriction Enzyme,They are synthesized by bacteria
to protect themselves from invading viruses.
class of endonucleases which cleaves DNA molecules at
specific nucleotide sequences called restriction sites,
1978 Nobel Prize,Arber,Smith & Nathans
Naming of enzymes
Over 150 different restriction endonucleases have been
identified.
Isolated from different bacteria
Recognize specific DNA sequences which are usually
4-8 bp long
Escherichia coli yields several different enzymes,one
is called EcoRI,The E is derived from the genus,and
the second and third letters (co) # from the species
name,The numbers refer to the strain,in this case
RY13.
Restriction Enzyme Specificity
e.g,Eco RI,GAATTC
Bam HI GGATCC,6-cutters”
Frequency,46 = average of 1 cut site per 4,096 bp
e.g,Sau 3A GATC,4-cutter”
Frequency,44 = average of 1 cut site per 256 bp
e.g,Not I GCGGCCGC,8-cutter”
Frequency,48 = average of 1 cut site per 65,536
Palindromes
Madam I’m Adam
madA m’I madaM
Blunt ends and sticky ends
,Blunt ends”
e.g,Sma I,Hae III,any other blunt-cutter
CCCGGG
GGGCCC
GGCC
CCGG
CCC
GGG
CC
GG
+ CCCCCGGGGG
Plasmid,Cloning Vectors
Origin of replication
Selectable marker
Cloning sites (unique)
Shuttle vectors
grow in more than one host
cell
Construction of Recombinant DNA Libraries
Genomic library:
A collection of clones representing the entire
chromosomal DNA sequence,These clones include
the entire gene,including introns (eukaryotes) and
regulatory regions.
cDNA library (complimentary DNA)
A collection of clones representing DNA copies of all
the different mRNA molecules in the cell,These
clones do not include the introns (eukaryotes) nor the
regulatory regions of the gene because they are
derived from mRNA.
Genomic Library Construction
Overlapping clones:
Random shearing
Partial digests,not all sites are cut
use 4-bp recognition (e.g.,Sau 3A) or rare-cutting enzymes
(notI; 8 bp recognition)
S S S S S S S S S S S S S S S S S S S S S
Genomic Libraries
Sometimes called clone banks or gene banks
To obtain a complete library the number of individual
clones required in a library is dependent upon:
size of the genome (the larger the more needed)
the size of the DNA insert in the cloning vector
(smaller insert sizes require more individuals)
Example,Human genome (3 billion bp) with an insert
size of 40,000 bps = 330,000 clones,But,if the insert
size is 200,000 bps,only 6,900 clones are needed to
represent the entire human genome,
Screening a library for a specific clone
NEED A PROBE!
Complementary DNA or RNA sequence
Use radioactive DNA to detect the
presence of the clone
fluorescent antibodies can also be used
to look for specific proteins
Isolating
mouse CF
gene with
human CF
probe
Characterization of cloned fragments
Construct a map of the fragment that shows the
location of all restriction endonuclease cut sites.
Determine the area within the clone that codes for a
specific gene.
Determine the sequence of nucleotides in the clone.
Genotyping with molecular biology
techniques
Genotyping with microsatellite
Detecting single base difference or SNP (single
nucleotide polymorphism)
RFLP,restriction fragment length polymorphism,
RFLP can arise from single base pair change and
used for its detection,RFLP can also arise from
other DNA changes (deletion or insertion),
SSCP,single strand conformation polymorphism
ASO,allele specific oligo
RAPD
RAPD,random amplification of polymorphic DNA
Used for isolating single-base-pair polymorphic
markers of unknown genome
