Biology 212 General Genetics
Lecture 14: Recombinant DNA II
Spring 2004
Reading: Chap. 11 pp. 408-420
Lecture Outline:
1. Vectors
2. Constructing a recombinant DNA
3. Genomic DNA and complementary DNA
Lecture:
To create a recombinant DNA you need:
 Sample of DNA
 A vector, usually a bacterial plasmid DNA or virus DNA that acts as a carrier of
the foreign gene
 Enzymes to cut DNAs (restriction enzymes) and rejoin DNAs (ligase)
Reasons for constructing recombinant DNA
 Isolate and purify new DNA sequence
 Prepare large amounts of DNA
 Produce large amounts of expressed proteins
1. Vectors
Properties of vectors:
 Replication origin, so it can replicate inside host cell
 Convenient restriction sites for cloning
 Genes that enable vectors and/or recombinants to be easily identified.
a. plasmids: extrachromosomal DNAs of bacteria
plasmid
chromosome
Examples of plasmid vectors
pBR322
and
pUC18
1
Feature
Origin of replication
Restriction sites
pBR322
Yes
A few
Antibiotic resistance genes
AmpR (ampicillin
resistance)
TetR (tetracycline
resistance)
pUC18
Yes
Many in a polylinker
(multiple cloning site)
AmpR
Genetic markers
Lac Z (gene for betagalactosidase)
b. bacteriophage lambda



virus that infects E. coli
contains 50 kb double stranded DNA genome in protein coat
vectors remove the middle region (a control region not required for virus
replication) and replace with foreign DNA
c. vectors for cloning large DNAs: human genome project
cosmids: plasmids containing lambda phage packaging gene
BACs: bacterial artificial chromosomes--modified fertility (F) plasmid vector
PACs: P1 artificial chromosomes--P1 virus (circular DNA) vector
YACs: yeast artificial chromosomes--contain centromere, telomeres, origin of replication
and foreign DNA
2. Constructing a recombinant DNA
a. digest DNA of interest with restriction enzyme
b. digest vector with same enzyme
c. mix DNAs together; sticky ends will associate
d. seal nicks with DNA ligase
e. use transformation to introduce recombinant DNA into bacteria
f. identify recombinants by antibiotic selection or by disruption of marker gene
Use of selective media to detect recombinants
Blue/white color screen for lacZ genetic marker
pUC18 vector
Lac Z gene: codes for enzyme beta-gelactosidase that cleaves lactose or other substrates.
Disruption of gene creates mutant that is unable to produce beta-galactosidase.
beta-galactosidase
X-gal (colorless substrate) ---- > blue color
2
LB ampicillin agar + X-gal
Recombinant (lacZ-)
Non-recombinant (lacZ+)
Screen for antibiotic resistance markers
pBR322
Insert foreign DNA into tetracycline resistance gene. Phenotype of bacteria with
recombinant DNA is then tetS (tetracycline sensitive).
Compare the growth of the transformed cells on LB ampicillin agar with or without
tetracycline
+ tetracycline
- tetracycline
Recombinant (ampRtetS)
Non-recombinant (ampRtetR)
Non-recombinant
3. Genomic DNA and complementary DNA
Prokaryotes and eukaryotes differ in the structure of most of their genes:
Feature
DNA in chromosomes
Gene packing
Gene structure
Prokaryotes
One circular DNA
Many genes, little noncoding DNA
Few genes with introns
Eukaryotes
Many linear DNAs
Much non-coding DNA,
genes far apart
Most genes with introns
Introns: Sequences of DNA inserted into genes that do not code for proteins
Bacterial gene:
gene (DNA)
transcription
5’ ~~~~AUG~~~~~~~~~~~~~~UGA~~~~~~3’ mRNA
translation
protein
3
Eukaryotic gene (Human, Fruit fly)
gene (DNA):
exon
intron
exon
intron
exon
transcription
5’ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~3’ 1º transcript
RNA processing
5’~~~~~~~~~~~~~~~~~~~~~~~~~~~3’ mRNA
translation
protein
Genomic DNA: all the DNA from an organism
cDNA: complementary DNA, a DNA copy of a messenger RNA
Many studies with recombinant DNAs in humans or higher eukaryotes are done with
cDNAs to simplify analysis.
 Most human DNA does not contain genes
 About 40,000 human genes/3 billion base pairs of genomic DNA
 Most of the genome contains repetitive DNA or DNA that does not code for
proteins
 The sequences of DNA that are transcribed to give mRNA are those that code for
proteins
 The cDNA can provide all the information about the parts of the gene that code
for protein and are most important for function
Preparation of cDNA:
a.
b.
c.
d.
e.
f.
isolate messenger RNA
add a primer
use reverse transcriptase and deoxynucleotide triphosphates to make a DNA copy
remove the RNA
use DNA polymerase I to synthesize the complementary DNA strand
cDNA can be cloned into a plasmid or phage vector or can be used for PCR
4
5' UGAGAUGCGCC………….AAAAAAA 3'
TTTTTTT 5'
mRNA
primer
+ reverse transcriptase
+ dATP, dCTP, dGTP, dTTP
5' UGAGAUGCGCC………….AAAAAAA 3'
3' ACTCTACGCGG…………..TTTTTTT 5'
5
mRNA
cDNA