Block II Lecture 1: Recombinant DNA Technology
Part I. DNA Manipulations: Basic Techniques
Overview of the Procedure
Cloning Vectors
Target Gene Selection and Acquisition
Restriction Endonucleases
Polymerase Chain Reaction (PCR)
DNA Ligation, Transformation, and Selection
Clone Identification and Screening
Restriction Digestion Analysis
Thermal Cycle DNA Sequencing
Library Construction and Analysis
Shotgun Approaches for Sequencing Genomic DNA
Cloning: To make identical copies
DNA cloning involves separating a specific gene or DNA segment
from a chromosome, attaching it to a DNA carrier molecule, and
replicating this modified DNA, thousands or millions of times,
through an increase in cell number and DNA copies per cell.
The result is selective purification and amplification of a particular
target gene or DNA segment from a complex mixture of DNA
molecules.
The methods used to accomplish these and related tasks are
collectively referred to as recombinant DNA technology or
genetic engineering.
Cloning vectors allow amplification of inserted DNA fragments
•Developed from
naturally occurring
bacterial plasmids
•Contain an origin of
replication (ori )
•Contain numerous
restriction sites
•Contain genes that
confer resistance to
antibiotics, thus
allowing selection of
bacterial colonies
carrying the plasmid
•Introduced into
competent bacterial
cells by transformation
Different types of cloning vectors
Plasmids: Circular DNA molecules which replicate separately
from the host chromosome. Plasmids used for genomic and
cDNA cloning. Bacterial host. Insert size range < 15kb.
Bacteriophage-based Cosmids: Linear DNA molecules
used for genomic and cDNA cloning. Bacterial host. Insert size
range < 20kb.
Bacterial Artificial Chromosomes (BACs): Circular DNA molecules
used for cloning very long segments of genomic DNA. Bacterial
host. Insert size range 100-300 kb.
Yeast Artificial Chromosomes (YACs) : Specialized DNA molecules
used for cloning very, very long segments of genomic DNA.
Yeast host. Insert size range 100-2000kb.
Mammalian expression vector
With the
exception
of budding
yeast,
plasmids are
uncommon
in eukaryotes.
Thus, most
eukaryotic
vectors are
based on DNA
or RNA viral
genomes.
Note : Multiple
Cloning Sites (MCS)
or a Polylinker Region
*
*
*
*
*
*
* Viral DNA sequences
Bacterial sequences
Restriction endonucleases cut DNA molecules at defined positions
A restriction enzymes binds to DNA at a specific sequence and make a double-stranded
cut at or near that sequence.
Blunt and sticky ends
Digestion of DNA
with different
restriction
endonucleases
5’ and 3’ overhangs
The same sticky ends produced by different enzymes
Polymerase Chain Reaction (PCR)
DNA from a selected region of the chromosome or genome can to be amplified a billion-fold,
effectively “purifying” it away from a complex mixture of DNA molecules.
Amplification of a DNA Segment
Long Product
REQUIREMENTS:
5’
Oligonucleotide primers which
flank the sequence of interest
A DNA Template (a few ng)
A thermal-stable
DNA Polymerase (TAQ)
5’
dNTPs
Long Product
An automated thermocycler
A repetitive three- step process : “Denature--Anneal--Elongate”
(94-97oC) (42-55oC) (72oC)
Polymerase Chain Reaction (PCR)
SP
LP
LP
SP
The Long
Product
(LP) acts as
template
for new
synthesis
Gives rise to
Short Product
(SP) whose
5’ and 3’ ends
are both set
by the primer
annealing
positions
Polymerase Chain Reaction (PCR)
SP
Sequential rounds
SP
In subsequent rounds, the
Short Products accumulate
in an exponential fashion
TET
TET
Following restriction digestion, the
vector and insert are purified by
agarose gel electrophoresis
DNA ligation reaction
is transformed into
“competent cells”
and then spread on
selective agar plates
Cut EcoRI / Pvu II /
Not I
DNA Marker
Cut EcoRI / Pvu II
Cut EcoRI / Pvu II
Cut EcoRI / Pvu II
DNA Marker
Insert
Distance
DNA Marker
Log10 bp
Analysis of Recombinant Clones: Restriction Enzyme Digestion
1.2% agarose gel cast
In 1X TAE buffer
DNA fragments stained
with ethidium bromide
and visualized by UV
illumination.
EcoR I
Pvu II
Not I
Vector
Insert
Analysis of Recombinant Clones: Thermal Cycle DNA Sequencing
dNTP
Base
PO4
O
HH
HH
OH H
ddNTP
Base
PO4
O
HH
H
HH
H
Genomic Library Construction using Bacteriophage l-based Vectors
The l genome contains “optional” DNA
* Cos site
*
*
Genes are arranged into functional groups
Insertion and Replacement Vectors
Cos sites incorporated into a plasmid = Cosmid
Insert size range < 20 kb
Genomic DNA Library Construction
Analysis: Colony Hybridization
Nytran or
Nitrocellulose
membrane
Add an in vitro
packaging mix
Shotgun Sequencing Approaches
A large segment of
genomic DNA or a
chromosome
A whole genome
Restriction digestion
Note:
A genomic map is needed to
provide a guide for sequencing
by showing the positions
of genes and other distinctive
features.
Closing a “sequencing gap”
Block II Lecture 1: Recombinant DNA Technology
Part II. Experimental Problems and Approaches
Assigning Genes to Chromosomal Locations
Genetic Mapping
RFLP and SSLP Analysis
Physical Mapping
Positional Cloning of a Target Gene
cDNA synthesis and expression cloning
Mapping Genes using ESTs
Cloning Large Multigene Families by Degenerate PCR
Cloning of a Target “Protein” and Physical Mapping
Genetic and Physical Mapping of a Gene to a Chromosome
Genetic mapping
enables physical
mapping
Genetic markers used
for chromosomal
mapping:
Restriction site
variation
Repetitive sequences
Genetic Linkage Analysis
Genetic Mapping
Restriction Fragment Length Polymorphism (RFLPs)
Useful molecular marker loci for chromosomal mapping and diagnosis of human
disease genes
This technique takes advantage of the ability of bacterial restriction enzymes to
cut DNA at specific target sequences that exist randomly in the DNA of other
organisms.
Generally, the target sites are found at the same position in the DNA of different
individuals within a population (i.e. the DNA of homologous chromosomes).
Frequently, a specific site is missing because of some silent mutation. The
mutation could be within a gene or a non coding intergenic region.
If an individual is heterozygous for the presence (+) and absence (+/ -) of a restriction
site, that locus can be used in mapping. The (+ / -) sites are detected by Southern
blot analysis using a probe derived from that region.
3 kb
Homolog 1
Homolog 2
Extent of probe
2 kb
1 kb
Southern blot
analysis of this
individual’s DNA
would detect three
fragments, 3, 2,
and 1kb in length.
3kb
2kb
1kb
Another individual might be homozygous for the long fragment and would show
only a 3 kb band on a Southern blot.
3 kb
Homolog 1
Homolog 2
3 kb
Extent of probe
Southern blot
analysis of this
individual’s DNA
would detect one
fragment 3kb in
length.
Multiple forms of this region constitute an RFLP
3kb
2 kb 1 kb
Homolog 1
Homolog 2
D
d
3 kb
In a cross of the two previous individuals, 50% of the progeny would show 3
fragments when probed, and the other 50% would show 1 fragment. This
result follows Mendel’s Law of Equal Segregation, just as a gene would.
3kb
2kb
1kb
3kb
2 kb 1 kb
Homolog 1
Homolog 2
D
d
3 kb
Hence, an RFLP can be mapped and treated like any other chromosomal site.
Linkage of the heterozygous RFLP to a heterozygous gene with D coupled to
the 1 plus 2 morph. Crossover between these sites would produce
recombinant products (D-3, d-2-1).
With this approach, the RFLP locus can be mapped relative to other
molecular markers.
“DNA Fingerprinting”used in modern forensics
Victim
Evidence
Suspect
Restriction Fragment
Length Polymorphism
(RFLP) Analysis
Simple-Sequence Length Polymorphisms (SSLPs)
D
VNTRs :
Variation in the
Number of
Tandem Repeats
or “Mini-satellite”
Molecular Markers
d
Probe binds repetitive
sequences
Restriction target sites are outside the repetitive array.
The basic unit of the array is indicated by the arrows.
The number of repeated units in a tandem array is variable. Individuals
heterozygous for different numbers of tandem repeats can be detected,
and the heterozygous site (s) used as a marker (s) for mapping.
This VNTR locus will form two bands on a Southern blot: one long and
one short. Similar to an RFLP locus, this heterozygous site can be used
for genetic mapping.
At present, VNTR analysis is rapidly performed using PCR.
Genetic profiling using Mini-Satellite VNTRs
VNTRs located on the short arm of Chromosome 6 were amplified by PCR.
The PCR Products were labeled with a blue or green fluorescent marker and
resolved on a polyacrylamide gel. Each lane displays the genetic profile of a
different individual. No two individuals will have the same genetic profile
because each person had a different set of mini-satellite variants, which give
rise to bands of different sizes after PCR. The red bands are DNA markers.
Positional cloning of a human target gene
Contigs
“Chromosomal Walking” technique used to identify single-disease genes in humans
cDNA Synthesis
DNA molecules copied from
an mRNA molecule by RT
and therefore lack introns in
genomic DNA
Isolate mRNA from
cell or tissue of
interest
Check integrity of
RNA prep on HCHO
gel
Convert total pool
of mRNA into cDNA
using RT
Clone cDNA into a DNA vector (e.g. l Zap) l to construct a
cDNA expression library. Propagate and amplify cDNA
library in a suitable host. Screen for cDNA of interest using
DNA probe or antibodies that recognize the encoded protein.
Gene Mapping using Expressed Sequence Tags (ESTs)
ESTs are obtained by sequencing
into the cDNA insert using a primer
based on the vector sequence
5’
3’
cDNA
EST DATABASE
A collection of partial cDNA sequences, generally 200 to 400 bp in length, that was generated
by sequencing vast numbers of cDNAs isolated from human cells and important model organisms
such as mouse, Drosophila, and Caenorhabditis elegans.
Composed of relatively short portions (tags) of genomic DNA sequences that are expressed in the
form of mRNA. The EST database is constantly updated as sequences from increasing number of
cDNA clones are added.
Genetic code contains redundancies = Degenerate
ATT-Ile
TAT- Tyr
TTA - Leu
STOP Codons
TAA
TAG
TGA
TTG- Leu
CTT- Leu
CTC- Leu
CTG- Leu
20 Different Naturally Occurring Amino Acids
64 CODONS : 61 encode amino acids
Computer programs apply
the triplet-based genetic
code to translate the EST
sequences into partial
amino acid sequence.
Three nucleotides (a codon)
are read from a specific
starting point. If a match is
found, then the EST
provides the unique DNA
sequence of that portion of
the cDNA.
A single probe that is
complementary to the
portion of the EST can
be used to screen a
genomic DNA library;
the probe could also be
used to screen a cDNA
library
NH2
tyr phe ile ser ser asn ser thr leu asn ala lys leu his leu thr
COOH
Odorant Receptors and the Organization of the Olfactory System
Cloning a
Large
Multi-Gene
Family by
Degenerate
PCR
Experimental design based on three assumptions:
1.
Odorant receptors likely belong to a superfamily of receptors
(i.e. seven transmembrane domain receptors) that transduce intracellular
signals by coupling to GTP-binding proteins
2.
The large number of structurally distinct odorous molecules suggests
that the odorant receptors themselves should exhibit significant diversity
and are likely to be encoded by a multigene family.
3.
Expression of odorant receptors should be restricted to the olfactory epithelium.
GOAL: To identify molecules in the olfactory epithelium that resemble members of the
seven transmembrane domain superfamily.
Step 1. Extract RNA from olfactory epithelium and prepare cDNA
Step 2. cDNA is amplified by PCR using a series of degenerate oligonucleotide primers
that anneal to conserved regions of members of the superfamily of G-coupled seven
transmembrane domain receptor genes.
5’ primers (match Domain II sequences)
II
VII
3’ primers (match Domain VII sequences)
Each of the five different 5’ primer was used in PCR
Reactions with each of six different 3’ primers.
Step 3. The amplification products of each PCR reaction were analyzed by agarose gel electrophoresis
Step 4. PCR products within the size range expected for this family of receptor (600-1300 bp) were selected
for further amplification with the appropriate primer pair to isolate individual bands. Each of the semi-purified
PCR products was digested with the restriction enzyme Hinfl and analyzed by gel electrophoresis.
(22 of the 64 PCR
products isolated)
PCR 13 yields a very large number of restriction fragments
whose molecular weight sums to a value 5- to 10-fold greater
than the original PCR product (13 different species of DNA)
Step 5. PCR 13 DNA was cloned into the plasmid vector Bluescript and 5 clones analyzed by DNA sequencing
Each clone exhibited a different DNA sequence,
BUT each encoded a protein that displayed
conserved features of the superfamily of seven
transmembrane receptor proteins.
The proteins encoded by all 5 clones shared
distinctive sequence motifs not found in other
superfamily members , indicating they were all
members of a NEW family of receptors
Step 6. Obtain full-length cDNA clones by screening cDNA libraries prepared from olfactory epithelium RNA
or RNA from enriched populations of olfactory sensory neurons
Primary screen used a mixture of PCR 13 DNA as the probe (20 positives)
Secondary screen used the original pair of primers used to amplify PCR 13 DNA (A4/B6)
Step 7. Confirm expression of isolated cDNAs is restricted to epithelium using Northern blot analysis
RESULT:
Identified 18 members of a novel, extremely large multi-gene family that encoded
olfactory receptors and lead to future work that merited the 2004 Nobel Prize
in Medicine.
Cloning of a “target protein X”
H2 N
H2 N
NH2
Digest with protease
COOH
H2 N
COOH
COOH
COOH
Step 1
H2 N
COOH
Protein X
Step 2
• Encoded by a pathogen
• Gene locus unassigned
Separate
peptides
COOH
Step 3
Automated peptide
sequencing
H2 N
Design a “degenerate” probe based on partial protein sequence
Mixture of 96 oligonucleotides
that encode a portion of the peptide
Degenerate
PCR
Note: If a Protein X EST database existed, you could design a single probe that
was based on partial protein sequences
Once DNA sequence of the target gene is available, you could:
• Map the entire gene and its location within the pathogen genome
• Clone and sequence the transcript(s) encoded by the Protein X gene
• Define the Protein X gene structure
• Construct expression plasmids for functional studies of Protein X in cells
• Mutagenize the Protein X cDNA using PCR-based site-directed mutagenesis and
perform structure-function analysis
• Produce recombinant protein X for vaccine development studies
REFERENCE MATERIALS FOR BLOCK 2/ LECTURE 1/ DNA Manipulations
Lehninger Principles of Biochemistry, 3rd edition, Chapter 29
An Introduction to Genetic Analysis , 7th edition, Chapters 6, 7, 12, and 13
(http://WWW.WHFREEMAN.COM/BIOLOGY)
FYI
Lab Math: A handbook of Measurements, Calculations, and Other
Quantitative Skills for Use at the Bench. D.S. Adams (2003) CSH
Laboratory Press.