MANIPULATION OF DNA AND GENE ISOLATION

1
UNIT 2 MANIPULATION OF DNA AND GENE
ISOLATION LECTURES 9. DNA Cloning and
Library Construction 10. Isolating Genes
2
9. DNA Cloning and Library Construction
a). DNA cloning i). Restriction
endonucleases ii). Cloning vectors iii). The
process of cloning a segment of DNA b). Library
construction i). Genomic libraries ii). cDNA
libraries
3
DNA mRNA
protein

• How does one isolate a gene for an inherited
  disorder?
• There are three options
• Start with a candidate protein
• DNA protein
• Start with a candidate mRNA
• DNA mRNA
• Direct positional cloning
• DNA
• All three options require the cloning of DNA.

4

• Restriction endonucleases
• Restriction enzymes cut DNA into specific
  fragments
• Restriction enzymes recognize specific base
  sequences in double-stranded
• DNA and cleave both strands of the duplex at
  specific places
• Characteristics of restriction enzymes
• 1. Cut DNA sequence-specifically
• 2. Bacterial enzymes hundreds are purified and
  available commercially
• 3. Restriction-modification system
• Bacteria have enzymes that will cleave foreign
  DNA hence, restrict the entry of viral
• DNA. To prevent the bacterias own DNA from
  being cut, there is a second enzyme that
• methylates the same sites recognized by the
  restriction enzyme (modifies that site).
• 4. Named (e.g., EcoRI) for bacterial genus,
  species, strain, and type
• 5. Recognize specific 4-8 bp sequences
• sequences have symmetry (they are palindromes)
• after cutting the DNA, the cut ends are either
• blunt
• staggered (overhangs) - cohesive ends facilitate
  cloning the DNA

5
4-base cutter cuts DNA into 256 bp
average-sized fragments in a random sequence
every 256 bp NO 256 bp average-size fragments
YES
Bar 256 bp
6
Products generated by restriction enzymes
COHESIVE ENDS EcoRI 5GAATTC3 5G A
ATTC3 3CTTAAG5 3CTTAA
G5 PstI 5CTGCAG3 5CTGCA
G3 3GACGTC5 3G ACGTC5
BLUNT ENDS HaeIII 5GGCC3 5GG
CC3 3CCGG5 3CC GG5
7
Formation of recombinant DNA molecules
cut DNAs
mix together fragments and anneal cohesive ends
seal 3, 5 ends by DNA ligase
recombinant DNAs
8
Vectors used in molecular cloning Vector
Insert (and host) Characteristics
size range Plasmid Small circular
DNA lt5 - 10 kb (bacteria, yeast) Bacteriopha
ge lambda Linear viral DNA up to 20
kb or phage lambda (bacteria) Cosmid
Hybrid of plasmid up to 50 kb (bacteria)
and phage Yeast artificial
DNA containing yeast 200 to 1000 kb chromosome
or YAC centromere, telomeres,
(yeast) and origins of replication
9

• Structure of pBR322 - a common cloning vector
• derived from a naturally occurring plasmid
• has antibiotic resistance genes for selection of
  
• transformants containing the plasmid
• has unique restriction enzyme cleavage sites for
• insertion of foreign DNA
• has origin of DNA replication (ori) for
  propagation in E. coli

gene for tetracycline resistance
gene for ampicillin resistance
EcoRI
Pst I
Sal I
10
Cloning a segment of DNA into a plasmid vector
PstI
Human DNA cut with PstI
P
pBR322 ampR, tetR
ampR
tetR
P
P
combine and ligate
P
tetR
pBR322 DNA cut with PstI inactivating the ampR
gene
tetR
pBR322 (human clone) tetR

• bacteria are transformed with the recombinant
  plasmid
• colonies that grow in tetracycline, but not in
  ampicillin are isolated

11

• Library construction
• two types of libraries
• a genomic library contains fragments of genomic
  DNA (genes)
• a cDNA library contains DNA copies of cellular
  mRNAs
• both types are usually cloned in bacteriophage
  vectors
• Construction of a genomic library

• vector DNA (bacteriophage lambda)
• lambda has a linear double-
• stranded DNA genome
• the left and right arms are essential
• for the phage replication cycle
• the internal fragment is dispensable

Bam HI sites
left arm
right arm
internal fragment (dispensable for phage growth)
12
human genomic DNA (isolated from
many cells)
NNG GATCCNN NNCCTAG GNN
Bam HI sites
cut with Bam HI (6-base cutter)
cut with Sau 3A (4-base cutter) which has ends
compatible with Bam HI
NNN GATCNNN NNNCTAG NNN
internal fragment
remove internal fragment
isolate 20 kb fragments
13
combine and treat with DNA ligase
package into bacteriophage and infect E.
coli
5
6
2
3
1
4

• genomic library of human DNA fragments
• in which each phage contains a different
• human DNA sequence

14
Partial restriction enzyme digestion allows
cloning of overlapping fragments
a contig

• isolation of 20 kb fragments provides optimally
• sized DNAs for cloning in bacteriophage
• partial digestion with a frequent-cutter (4-base
  cutter) allows production
• of overlapping fragments, since not every site
  is cut
• overlapping fragments insures that all sequences
  in the genome are cloned
• overlapping fragments allows larger physical
  maps to be constructed as
• contiguous chromosomal regions (contigs) are put
  together from
• the sequence data
• number of clones needed to fully represent the
  human genome (3 X 109 bp)
• assuming 20 kb fragments
• theoretical minimum 150,000
• 99 probability that every sequence is
  represented 800,000

15
All possible sites
Results of a partial digestion
uncut
cut
16

• Construction of a cDNA library
• reverse transcriptase makes a DNA copy of an RNA

The life cycle of a retrovirus depends on reverse
transcriptase
retrovirus
2. the capsid is uncoated, releasing genomic RNA
and reverse transcriptase
3. reverse transcriptase makes a DNA copy
1. virus enters cell and looses envelope
4. then copies the DNA strand to make it
double-stranded DNA, removing the RNA with RNase H
6. it is translated into viral proteins, and
assembled into new virus particles
5. the DNA is then integrated into the host cell
genome where it is transcribed by host RNA
polymerase II
new viruses
17

• cDNA library construction

AAAAA
5
3 mRNA (all mRNAs in cell)
anneal oligo(dT) primers of 12-18 bases in length
AAAAA TTTTT
5
3 5
3
add reverse transcriptase and dNTPs
AAAAA TTTTT
5 3
3 5 cDNA
add RNaseH (specific for the RNA strand of an
RNA-DNA hybrid) and carry out a partial
digestion
AA TTTTT
5
3
short RNA fragments serve as primers for
second strand synthesis using DNA polymerase I
18
AAAAA TTTTT
5 3
short RNA fragments serve as primers for
second strand synthesis using DNA polymerase I
DNA ligase seals the gaps
AAAAA TTTTT
5 3
double-stranded cDNA
19
AAAAA TTTTT
5 3
NNNNNNNNG NNNNNNNNCTTAA
EcoRI linkers are
ligated to both ends using DNA ligase
AAAAANNNNNNNNG TTTTTNNNNNNNNCTTAA
5 3
AATTCNNNNNNNN GNNNNNNNN

• double-stranded cDNA copies of mRNA with EcoRI
  cohesive ends are
• now ready to ligate into a
  bacteriophage lambda vector cut with EcoRI

20
EcoRI sites
cDNAs
combine cDNAs with lambda arms and treat
with DNA ligase
package into bacteriophage and infect E.
coli
5
6
2
3
1
4

• cDNA library in which each phage contains
• a different human cDNA