10. DNA Cloning and Isolating Genes

1
10. DNA Cloning and Isolating Genes
a). DNA cloning i). Restriction endonucleases
ii). Cloning vectors iii). The process of clo
ning a segment of DNA b). Library construction
i). Genomic libraries ii). cDNA libraries
c). Library screening plaque hybridization
d). Probes for library screening
i). Starting with a protein synthetic oligonuc
leotide antibody ii). Starting with mRNA d
ifferential cDNA library screening
expression screening
2

• Learning Objectives for Lecture 10
• Understand the purpose for cloning DNA
• Understand the characteristics of restriction
  enzymes and how they are useful for DNA cloning
  
  
• Understand what cloning vectors are and how they
  are used to carry and amplify DNA inserts
  
• Understand the basic differences between genomic
  and cDNA libraries
  
• Understand how genomic libraries are constructed
  
  
• Understand the purpose for having overlapping
  DNA fragments in genomic libraries and how they
  are generated
  
• Understand how cDNA libraries are constructed
  and the use of reverse transcriptase for their
  construction
  
• Understand the rationale for library screening
• Understand the method of plaque hybridization
• Understand the four methods for library
  screening and when they are put into use

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-siz
ed fragments in a random sequence
every 256 bp NO 256 bp average-size fragment
s YES
Bar 256 bp
6
Products generated by restriction enzymes
COHESIVE ENDS EcoRI 5GAATTC3 5G
AATTC3 3CTTAAG5 3CTTAA G5 P
stI 5CTGCAG3 5CTGCA G3
3GACGTC5 3G ACGTC5 BLUNT E
NDS HaeIII 5GGCC3 5GG CC3 3CCG
G5 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) Ch
aracteristics size range Plasmid S
mall circular DNA
(bacteria, yeast) Bacteriophage 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 ce
ntromere, 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 ge
ne
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
left arm
right arm
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 wi
th 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 polym
erase 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 dige
stion
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
AAA TTTTT
5 3
DNA polymerase I removes the remaining RNA with
its 5 to 3 exonuclease activity and continues
synthesis
AAA TTTTT
5 3
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
left arm
right arm
package into bacteriophage and infect E.
coli
5
6
2
3
1
4

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

21

• Plaque hybridization
• This is a general technique required for a
  number of
  
• specific approaches for isolating cDNA
  or genomic clones
  
• Generally, one starts by
• 1). Isolating a cDNA sequence from a cDNA
  library, then
  
• 2). The gene from a genomic library using the
  cDNA as a probe
  
• Information gained from cDNA and genomic clones
• 1). cDNA clones provide the amino acid sequence
  of the
  
• full-length protein, unencumbered by intron
  sequences
  
• 2). Genomic clones provide the control regions
  and are
  
• required for searching for mutations
• Library screening four experimental approaches
• Starting with a protein
• 1). Synthetic oligonucleotide - plaque
  hybridization
  
• 2). Antibody - variation of plaque
  hybridization
  
• Starting with mRNA

22

• Library screening
• plaque hybridization
• plate phage library on lawn of E. coli (bacteria
  phage)
  
• plaques form as a consequence of a spreading
  lytic infection
  
• starting with a single phage-infected bacterial
  cell
  
• each phage plaque is a clone of identical
  recombinant phage
  
• prepare replica of phage plaques and hybridize
  DNA with probe

E. coli lawn is grown on agar plate and then
overlayered with the recombinant phage library.
Wherever a single bacteriophage particle
infects a bacteria cell, a plaque will form.
This is a clear area caused by the lysis
of bacteria on the lawn of E. coli.
A replica of the agar plate is made on a
nitrocellulose sheet - the DNA is denatured
and adheres to the nitrocellulose.
The nitrocellulose is hybridized with a labeled
DNA probe (such as an oligonucleotide) and
the nitrocellulose is exposed to X-ray film.
X-ray film
spot on film indicates a plaque containing DNA of
interest
23
(No Transcript)
24

• How does one isolate a gene for an inherited
  disorder?
  
• Start with a candidate protein
• DNA protein
• If a protein candidate has been identified for a
  genetic
  
• disease it can be used to make a probe to screen
  for the gene
  
• 1. oligonucleotide probe
• purify the protein of interest
• partially sequence the protein
• find a region having amino acids with the fewest
  possible codons
  
• predict a DNA sequence that could represent a
  gene region
  
• encoding a portion of the protein
• synthesize a set of degenerate oligonucleotides
  for that region
  
• hybridize the labeled oligonucleotide to the
  phage library

MET.GLU.PHE.TYR.ILE.CYS.GLN.LYS amino acids
AUG.GAA.UUU.UAU.AUU.UGU.CAA.AAA
G C C C C G G all possible
A
oligonucleotides 1 X 2 X 2 X 2 X 3 X 2 X 2 X 2
192-fold degenerate

25

• 2. antibody probe
• purify the protein of interest
• make an antibody to that protein
• construct cDNA library to express recombinant
  proteins in E. coli
  
• use the antibody to detect the protein being
  made from the cloned
  
• cDNA encoded by the recombinant phage in E.
  coli using plaque
  
• hybridization method modified for antibody
  probes

bacterial promoter and Shine-Dalgarno sequence
left arm
right arm
human cDNA insert
26

• How does one isolate a gene for an inherited
  disorder?
  
• Start with a candidate mRNA
• DNA mRNA
• mRNA candidates can be identified by comparing
  mRNA
  
• populations between normal and abnormal tissues,
  or by
  
• looking for a specific function encoded by the
  mRNA
  
• 1. differential cDNA library screening
• prepare duplicate plaque replica plates
• hybridize one with a labeled cDNA probe made to
  all the mRNAs
  
• in the normal cell and hybridize the other
  (duplicate) with the
  
• corresponding probe to the abnormal cell
• differences in cell function should be reflected
  by differences
  
• in the mRNA populations
• any plaques showing differential hybridization
  are candidates

no hybrid- ization to this plaque
differentially expressed clone
hybridization with cDNA probe from nor
mal cells
hybridization with cDNA probe from abnorma
l cells
27

• 2. expression screening
• develop a cell-based functional assay for the
  abnormality
  
• (e.g., a transport assay)
• construct cDNA library in a way that will allow
  expression of
  
• protein in mammalian cells
• inject groups of cDNA clones into cells and
  assay function
  
• narrow down cDNA clones using smaller groups of
  clones until
  
• the function is observed with a single cDNA
  species

• inject groups of cDNA clones
• if the function being assayed is
• observed, divide the group of clones
• into smaller groups and retest
• continue process of testing smaller
• groups until the function being
• assayed is obtained with one clone

• for example, inject clones
• and test cells for transport activity

left arm
right arm
human cDNA insert
mammalian promoter
28
-

-
-
-
-
-


-
-
104
102
-
-
-
-
-