Download BNS216 - Staff

Basis of course
•
•
•
•
Understand the technology
Understand the terminology
Gain some practical experience
The applications in biotechnology and basic biology
next year
• Why?
– Fluidity next year (don’t need to explain terms and
technology while discussing the applications)
– Shows what molecular biology projects are like
• Disadvantage
– Technology dominated. Can make it a bit boring
Lectures
• Two per week, however
– The Tuesday lecture may be used to
discuss data from practicals
– In some lecture slots the in course test will
be held
– Approximately formal 15 lectures
Practicals
• I will supervise the first two, Dianne
Ford the second two
• Objectives of practicals
– Get used to pipetting small quantities
– Reinforce lectures
– Help focus on project choice for nexy year
– Schedules soon
BNS216
• Phases of course
– Isolation of a specific DNA sequence (gene
or cDNA)
– Analysis of isolated DNA sequence
• DNA sequencing
– Manipulation of DNA sequence
• PCR to introduce restriction enzyme site
• PCR to change codon
• PCR to detect specific DNA sequence
BNS216
– Production of recombinant protein
• Expression vectors
– Detecting genes and transcripts
• Northern hybridization
• Southern hybridization
• RTPCR
– Manipulation of eukaryotic organisms
BNS216
• Isolation of a gene
– Construction of a gene library
•
•
•
•
•
Choice or organism
Restriction enzymes
DNA ligase
Vectors and which ones to use
Screening startegies
– Prokaryotic and you look for the protein product
– Eukaryotic and you look for the DNA
BNS216
• Isolation of a cDNA
– Purification of mRNA
– DNA from mRNA
– Construction cDNA library
– Screening the library Analysis of DNA
• DNA sequencing
• Why you need to know the sequence
• Automated method with fluorescent
dideoxynucleotides
BNS216
• Manipulation of DNA
– PCR and how it relies on knowing the
sequence
• Introduction of restriction enzyme sites
• Changing the amino acid sequence of a protein
• Expression of proteins in bacteria
• Expression vectors
– How they enable foreign genes or cDNAs
to be expressed
– How expression vectors are regulated
BNS216
• Detecting transcription and genes
– Northern hybridization
– RTPCR
– Southern hybridization
• Manipulation of eukaryotic multicellular
organisms
– Transgenic animals
• Insertion of foreign genes by microinjection
• Inactivating genes by homologous
recombination in stem cels
– Transgenic plants
Assessment
• Exam:
– 60 % of module
– Answer 3 questions from 5
– Must get >35 % to pass module
• Practicals
– 20 % of module
– Four practicals starting on 4th February
Assessment
• In course tests
– Four tests
• Test one: Isolation of a gene or cDNA
• Test two: DNA sequencing and PCR
• Test three: Expression vectors and nucleic acid
detection
• Test four: Manipulating eukaryotic organisms
BNS216 references
• Difficult!
– Gene Cloning: An introduction T.A. Brown
OK quite simple but not in same way I
teach it
– Principles of Gene Manipulation: An
introduction to genetic engineering
R.W. Old and S.B. Primrose. Quite
detailed, some of which is unnecessary
– Use any standard molecular biology or
genetics text book, there will a section on
BNS216
What is genetic engineering or
recombinant DNA technology?
• A suite of technologies that enable you
to
• Isolate and characterise genes
• Produce and characterise proteins
• Alter the genetic make up of an
organism
– New genes
– Loss of existing genes
Applications of genetic
engineering
• Basic understanding of biology
– Protein structure and function
– Regulation of gene expression
– Importance of proteins in whole organisms
(gene knockouts or null mutations)
Applications of genetic
engineering
• Practical applications
– Production of industrially important proteins
– Change the properties of proteins
– Modification of the phenotype of whole
organisms
– Diagnosis
– Primary applications in medicine and
agriculture
– Others include chemical, paper and
detergent industries
Applications
• More details on applications
• Protein production
• Pharmaceutical proteins.
– Constant supply and safe
– Growth hormone, insulin, Factor VIII and
IX, antitrypsin
Proteins
• Microbial proteins
• Microbes grow poorly but produce
valuable enzymes
– Hyperthermophiles
– Anaerobes
– Archaebacteria
• Genetic engineering makes these
proteins available to industry
Enzymes used in the Food
Industry
• Glucose isomerase (Food industry)
• Xylanases (Paper industry)
• Cellulases (Energy and detergent
industries)
• Phytases (animal feed)
• Protein engineering
– Rational design
– Forced protein evolution
Modifying organisms
• Genetically engineered foods
– Herbicide resistance
– Pesticide resistance
• Good for consumer, farmer or biotech.
Companies?
– Golden rice with increased vitamin A and
oil seed rape with better polyunsaturated
fats
• Good for consumer?
Genetically engineered foods
• Risks
– The environment, spread of resistant
weeds, alter ecological balance?
– Human health. Will we get increased
antibiotic resistance
– Will the transgene be deleterious to
human health?
Change phenotype of farm
animals
• Convert them into bioreactors to
produce pharmaceutical proteins. Why?
• Change their biochemistry so
• More efficient use of nutrients
• Better quality end-products e.g. milk
and meat
– Humanising milk
– Increase polyunsaturates
Change phenotype of small
animals
• Generate animals for human disease
influenced by diet
• Colon cancer
• nvCJD
• Heart disease
Gene therapy
• Correct genetic defect
– Not in germ line
– Not transmissible
Diagnosis
• Diagnosis
– Human genome sequenced
– Identify all genes soon
– Immediate diagnosis test
• Hungtintons
• Muscular dystrophy
• Cystic fibrosis
• Sickle cell anaemia
• Alzeihmer
• Breast cancer
• Colon cancer
• Heart disease
– Good or bad?
Diagnosis
• Reduce incidence of disease
– Pregnancy termination
– Pre-implantation selection
• Start treatment to prevent disease
– Prophylactic mastectomy
– Colon removal
– Physiotherapy
• Stress if diagnosed. Do you want to
know?
• Insurance and job prospects?
Genetic engineering history
• Pioneered by Cohen and Boyer 19721974 (bacterial systems)
• Southern hybridization 1975
• DNA sequencing 1977-1980
• Transgenic animals 1980
• Polymerase chain reaction 1985
• Site-directed mutagenesis 1985
Where do we start?
• If we want to do genetic engineering
how do we start?
• Isolate the gene of interest
– Select organism containing gene
– Construct a gene library
– Select members of the gene library that
contain the gene of interest
How do you start doing recombinant
DNA technology?
• Isolate the gene of interest
• Lets isolate (clone) a cellulase gene
– Identify organism that contains the gene
•
•
•
•
•
Rumen
compost
Soil
Leaf litter
Decaying wood
Isolating a cellulase gene
Isolate chromosomal
DNA
Fragment DNA
Mix and ligate
Vector
E. coli
Transform
Gene library
Properties of vector DNA
•
•
•
•
Replicates in bacterium
Foreign DNA inserted will be stable
Normally extra-chromosomal
Easy to select bacterium containing
vector (confers antibiotic resistance)
• Vectors
– Plasmid (extra chromosomal circular DNA)
– Bacteriophage
– Cosmids
– Artificial chromosomes
Gene library
Screen library
for appropriate gene
(cellulase gene)
Isolate plasmid
Isolating a cellulase gene
Isolate chromosomal
DNA
Fragment DNA
Restriction endonucleases
• Enzymes that cut DNA at specific
sequences
• Discovered in the early 1950s
• Agent that enables bacteria to be
immune to bacteriophage
• Host-controlled restriction
• Mainly found in bacteria
• Over 1200 characterised
Restriction endonucleases
• Three types only Type II important in
genetic engineering as they cut the
sequence they recognise
• Target sequences generally palindromic
• Recognise 4, 6 or 8 nucleotides
Restriction endonucleases
GAATTC
CTTAAG
EcoRI
CTGCAG
GACGTC
PstI
CCCGGG
GGGCCC
SmaI
G
AATTC
CTTAA
G
Sticky ends
CTGCA
G
G
ACGTC
CCC
GGG
GGG
CCC
Blunt ends
Restriction endonucleases
• Named after organism
• e.g. EcoRI = Escherichia (E) coli (co) strain R
(R). I refers to Ist enzyme isolated from
organism
• Why doesn’t a bacterial restriction
endonuclease digest its own DNA?
• The bacterium produces a DNA methylase
that recognises same sequence as restriction
endonuclease
• Methylates target DNA sequence which
makes it resistant to endonuclease cleavage
Restriction enzymes and DNA methylase
Foreign DNA
GAATTC
CTTAAG
EcoRI
G
AATTC
CTTAA
G
Host DNA
GAATTC
CTTAAG
DNA methylase
CH3
GAATTC
CTTAAG
CH3
EcoRI
CH3
GAATTC
CTTAAG
CH3
Mix and ligate
Vector
E. coli
Transform
Gene library
Forming hybrid or recombinant DNA molecules
using restriction enzymes and DNA ligase
GAATTC
CTTAAG
GAATTC
CTTAAG
Digest with EcoRI
G
AATTC
CTTAA
G
G
AATTC
CTTAA
G
Mix DNA
DNA ligase
DNA ligase
GAATTC
CTTAAG
GAATTC
CTTAAG
GAATTC
CTTAAG
GAATTC
CTTAAG
GAATTC
CTTAAG
Recombinant or hybrid DNA
GAATTC
CTTAAG
Inserting chromosomal DNA into a vector
Chromosome
Vector
GAATTC
CTTAAG
GAATTC
CTTAAG
GAATTC
CTTAAG
Cut with EcoRI and add DNA ligase
Recombinant vector
GAATTC
CTTAAG
GAATTC
CTTAAG
More details on each stage
• Chromosomal DNA is only partially cut
because?
• Don’t know if the restriction enzyme
cuts in the gene
• Plasmid vector is designed to enable
selection for recombinant plasmid
– pUC or pBluescript-based plasmid vectors
– Contains two selection genes ampicillin
(antibiotic) and LacZ; codes for galactosidase
pUC18
HindIII
EcoRI
BamHI
Cells containing
pBluescript are
ampicillin resistance
and blue on X-Gal
Origin of replication
LacZ’ encodes -galactosidase
Ampr confers ampicillin resistance
Bromo-chloro-indoyl--galactopyranosidase or X-Gal (Clear)
-galactosidase
Bromo-chloro-indoyl (Deep blue insoluble)
+
galactose
Inserting chromosomal DNA into a vector
Chromosome
GAATTC
CTTAAG
Vector
GAATTC
CTTAAG
GAATTC
CTTAAG
Cut with EcoRI and add DNA ligase
Recombinant vector
GAATTC
CTTAAG
GAATTC
CTTAAG
Ampicillin resistant; -galactosidase negative (White on X-Gal)
LacZ gene codes for -galactosidase
Ampicillin resistance gene
Wild type vector
GAATTC
CTTAAG
Ampicillin resistant; -galactosidase active (Blue on X-Gal)
LacZ gene codes for -galactosidase
Ampicillin resistance gene
E. coli sensitive
to ampicillin
Ampicillin resistant;
-galactosidase active (Blue on X-Gal)
Ampicillin resistant; -galactosidase
negative (White on X-Gal)
Bacteria from ligation plated
on ampicillin and X-Gal
Contains
wild type
plasmd
Contains
recombinant
plasmd
Gene library
• Collection of microbes (e.g. Escherichia coli)
each one containing a recombinant vector
• Each recombinant vector contains a random
region of the target chromosome
• The number of microbes in the library is large
• Thus any gene in the target organism’s
genome is present in at least one member of
the gene library
Mix and ligate
Vector
E. coli
Transform
Gene library
Size of gene library
N = ln(1-P)
ln (1-A/B)
N = Number of clones
P = 95 % probability of finding gene
A = Average size of DNA fragments
B = Total size of genome
E. coli has genome of 4,800,000 nucleotides
Average size of insert is 10,000 nucleotides
Number of clones for 95 % probability is 1700
Size of gene library
• If genome is large e.g. human genome
(3 x 109) then number of clones to make
library becomes unrealistic (1058000) if
using a plasmid vector (accepts only 10
kb as larger DNA can’t be transformed)
• Therefore need to use vectors that can
accept larger pieces of DNA
– I.e. if each vector contains a large piece of
DNA you don’t need so many clones to
make a gene library
Vectors that accept larger
DNA
•
•
•
•
Plasmid: 10 kb
Lambda bacteriophage: 18-25 kb
Cosmid: ~40 kb
Yeast or bacterial artificial chromosome:
100-1000 kb
Gene library
Screen library
for appropriate gene
(cellulase gene)
Isolate plasmid
Screening gene library for
cellulase gene
• Assume bacterial genes will express in
Escherichia coli
• Escherichia coli does not degrade
polysaccharides
• Screen library by looking for members
that degrade cellulose
• Similar approach for other
polysaccharidases (amylases,
pectinases, xylanases etc)
Vectors
• Lambda vector
• Infects E. coli replicates and then
viruses released
• End of genome are 12 bp sequences
known as cos sequences.
• Cos sequences play an important role in
packaging viral DNA into capsids (head
of the virus)
Lambda infects E. coli
DNA injected
into E. coli
DNA replication generating
concatamers
Lambda DNA is linear in virus
Cos sequence is 12 nucleotides and single stranded
The two cos sequences are complementary
cos sequences hybridise in E.coli
to form circular genome
cos
lambda DNA cos etc etc
Replicates by rolling ciricle in E. coli
to produce concatemers
Lambda head genes transcribed and translated
to produce head proteins
Endoglucanase A expressed. Cuts DNA at cos sequence
and assists packaging lambda DNA into viral capsid
(head proteins)
Endoglucanase A cuts at cos
sequence
Tail genes then expressed
Tail bind to heads to form virus
Lambda virus produces lysozyme that hydrolyses
bacterial cell wall releasing viruses to attack
other bacterial cells
Lambda infects E. coli
DNA injected
into E. coli
DNA replication generating
concatamers
Replication by a mechanism called rolling circle
Vectors
• More information on vectors
• Lambda vector
• Libraries contain larger inserts than
plasmids (20-25 kb)
• Naked Lambda DNA can’t be
transformed into E. coli
• Lambda DNA can be packaged into a
virus
• Virus then infected into E. coli
In vitro packaging
Two E. coli mutants. One synthesises the  tails the other the
 heads and A protein
1. Take DNA and mix with E. coli extracts containing
 heads and A protein
Cos sequence
Internal DNA
+
2. Packaged DNA is then mixed with E. coli extracts
containing  tails
3. The virulent phage can then be used to infect E.coli to
form plaques in a lawn of bacteria
Lambda vector for genomic
cloning
1.Lambda genome is 40 kb
2. Lambda vectors contain right arm, left arm and
central region. At ends are single stranded cos sequences
3. Genes in central region not essential
4. Restriction sites at boundary of central region
and the two arms.
5. Used to clone DNA about 20 kb
Cloning DNA into vectors
cos
Left arm
Central Right arm
Cut out 3 regions
and purify arms
Chromosomal DNA
Cut chromosome with
same enzyme as lambda
Mix DNA and add
DNA ligase
DNA
will form
concatamers
Will not package
Recombinant lambda will
package. Why?
DNA packaging is size dependent
Endonuclease A
cleaves cos sequence
DNA too small
to package <18 kb
cos
DNA too large to
package >25 kb
DNA packages
18-25 kb
Cosmid vectors
• Problem with
vectors is that you
can’t transform large
pieces of DNA into
E. coli
• Cosmid
Cosmid vector
Cos
Ampr
– Similar to plasmid
• Ampicillin resistance
gene
• 5 kb in size
• Unique BamHI site
• Cos sequence
Origin
BamHI
Cosmid vectors
• Accepts as much as 40 kb of
chromosomal DNA
• Why?
– Smuggle cosmid into E. coli by packaging
it into lambda virus as it has a cos
sequence
Cosmid vector
Cos
GGATCC
CCTAGG
GGATCC
CCTAGG
Ampr
Cut with BamHI
Cut with BamHI
Origin
BamHI
GATCC
G
GATCC
G
G
CCTAG
G
CCTAG
Mix DNA at very
high concentration
and add DNA ligase
GGATCC
GGATCC
GGATCC
GGATCC
GGATCC
GGATCC
GGTACC
GGATCC
Packaged in vitro
Lambda particle
injects cosmid in E. coli.
E. coli is viable as
no lambda genes in
cosmid so it acts as
a normal plasmid
How do you select
for E. coli cells containing
cosmids?
They are resistant
to ampicillin
Telomere
Centromere
Telomere
Origins of
replication
Accepts up to 1 Mb of chromosomal DNA
Yeast Artificial Chromosome (YAC)
Accepts up to 1 Mb of DNA
centromere
origin
of replication
sup4 gene
SnaBI site
Trp1
gene
BamHI sites
Ura3 gene
Telomeres
Yeast Artificial Chromosome
Chromosomal DNA
centromere
origin
of replication
sup4 gene
Ura3 gene
SnaBI site
Telomeres
Trp1
gene
BamHI sites
Cut with SnaBI
Mix and add
DNA ligase
Cut with
SnaBI and
BamHI
Telomere
Trp1
Ori
Cent
sup4’
0.1-1 Mb
chromosomal DNA sup4’ Ura3 Telomere
Transform recombinant YACs into mutant yeast
that lack Ura3 and Trp1 genes (I.e. can’t make tryptophan
and uracil) so the amino acid and nucleotide must
be added for yeast to grow
Transformed yeast plated on media lacking tryptophan and uracil
Colony contains
recombinant YAC
Colony contains
wild type YAC
Plate yeast on medium lacking uracil and
tryptophan. Yeast colonies that grow contain YAC
and those that are pink contain recombinant YAC
as this indicates inactivation of Sup 4
What vectors for what
libraries?
Vector
Plasmid
Insert
size
<10 kb
Lambda phage 18-25 kb
Cosmid
YAC etc
What libraries
Bacteria
Yeast
34-45 kb
Intermediate
eukaryotes
0.1 – 1 Mb Higher Eukaryotes
Human library requires >1,000,000 plasmid clones
Human library requires 14000 YAC clones
Screening bacterial gene
library for a specific gene
• Assume bacterial genes will express in
Escherichia coli
• Screen for gene by looking for protein
that
– Changes phenotype of E. coli
• e.g. confers ability to degrade a polysaccharide
(cellulase, xylanase etc) confers green
fluorescence (green fluorescent protein)
– Changes phenotype of an E. coli mutant. Known
as complementation
– Screen for protein directly using an antibody
Complementation
• Isolate gene from a bacterium that E.
coli contains
– e.g. Isolate B. subtilis gene coding 1st
enzyme in leucine biosynthsis pathway
• Assume bacteria have same pathway
for leucine synthesis
• Step 1: Isolate a mutant of E. coli
unable to synthesise leucine
– I.e. gene for 1st enzyme non-functional
thus enzyme not produced
Complementation
(generating a leucine auxotroph)
Add mutagen
E. coli culture
Plate out on
media containing
leucine
Mutated E.coli culture
Colony 4 requires
leucine for growth
(leucine auxotroph)
Transfer
colonies
to media
lacking leucine
Complementation
• Leucine auxotrophs isolated require
leucine for growth
– Defect in gene coding for leucine synthesis
• Which gene in pathway mutated?
• Assay leucine auxotrophs for mutant
lacking enzyme one
Complementation
• Construct a gene library of Bacillus
subtilis chromosomal DNA
– Digest chromosome
– Insert into E. coli vector to make a library of
recombinant vectors
– Transform these recombinant vectors into
the E. coli leucine auxotroph lacking
enzyme 1
– Plate out library on media lacking leucine
Complementation
• E. coli colony that grows contains
recombinant plasmid with Bacillus
leucine biosynthetic gene that codes for
1st enzyme in leucine synthesis
• The recombinant plasmid has overcome
or complemented the E. coli mutation
Antibody screening
• Some bacterial genes code for proteins
that don’t change phenotype of E. coli
wild type or mutant e.g. asparaginase
from Erwinia
• Erwinia asparaginase converts
asparagine to aspartic acid
• Used as a major ingredient of
chemotherapy of childhood acute
leukaemia
Antibody screening
• Porton needed to produce more to
supply world demand
– Overexpress enzyme in E. coli
• How do you isolate gene?
• Make a gene library of Erwinia DNA in
E. coli
• Screen library using antibody specific to
Erwinia asparaginase
• How do you produce antibody?
Antibody Production
Erwinia
Purify asparaginase
from Erwinia
Blood of rabbit
contains antibodies
to Erwinia asparaginase but
no other Erwinia protein
Screening E. coli library of Erwinia DNA
1
2
3
4
5
6
7
8
9
Lyse bacteria with lysozyme
Protein binds to filter and
thus a print of the bacterium’s
proteins replaces colony
1
2
3
4
5
6
7
8
Plate out E. coli
cells on nylon filter
9
Add inert protein
(e.g. bovine serum
albumin) to block
protein binding sites
on nylon filter
1
2
3
4
5
6
7
8
9
Screening E. coli library of Erwinia DNA
1
2
3
4
5
6
7
8
Add antibodies
labelled with
radioactive iodine
9
www.staff.ac.ncl/h.j.gilbert
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Expose filter to X-ray film
to detect where antibody
has bound (radioactive and
thus blackens X-ray film)
Practical information
• Experiment 1: Each phage has an insert of
approximately 20 kb
• The fact that the cellulase gene is 1 kb not
really relevant
Screening a eukaryotic gene
library
• Will the gene express?
– No, lack of promoter, ribosome binding
sequence and introns
• Use nucleic acid hybridisation
Nucleic acid hybridization
1
2
3
4
5
6
7
8
NaOH added to lyse bacteria
1
and denature DNA into
single strand. Filter is
heated to 70 to immobilise
4
single strand DNA
7
9
2
3
5
6
8
9
Single stranded print of
bacterium’s DNA instead
of a bacterial colony
Plate out E. coli
cells on nylon filter
1
2
3
4
5
6
7
8
9
Inert DNA to block
vacant DNA binding
sites on filter
Nucleic acid hybridization
1
2
4
5
7
8
3
Add radioactive
nucleic acid probe
that binds to target
gene
6
9
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Expose filter to X-ray film
to detect where nucleic acid
has bound (radioactive and
thus blackens X-ray film)
Screening a eukaryotic gene
library
• Where does the probe come from?
– Homologous gene from other organism
– Oligonucleotide based on protein
sequence or known sequence of
homologous gene
– Differential screen (deal with this later;
microarray analysis)
Screening a eukaryotic gene
library
• Homologous gene from other organism
– Mammalian genes are very similar
– Thus if trying to get human gene screen
with the equivalent gene from another
organism
– Oligonucleotide based on protein
sequence or known sequence of
homologous gene
– Purify protein and determine sequence
– Build a nucleotide sequence which codes
for protein sequence
Amino acid sequence
Met-Asn-Lys-Trp-Glu-Met
Met = ATG; Asn = AAT or AAC; Lys = AAA or AAG;
Trp = TGG; Glu = GAA or GAG
How many probes must we make?
ATG AAT AAA TGG GAA ATG
ATG AAT AAA TGG GAG ATG
ATG AAT AAG TGG GAA ATG
ATG AAT AAG TGG GAG ATG
ATG AAC AAA TGG GAA ATG
ATG AAC AAA TGG GAG ATG
ATG AAC AAG TGG GAA ATG
ATG AAC AAG TGG GAG ATG
1st Test in BNS216
• Test is next Thursday February 26th
• One hour replaces lecture
• Multiple choice and single word or
simple diagram answers
• Consists of
– Construction and screening of gene
libraries
Construction of gene libraries
• Gene library
– Restriction endonucleases
– DNA ligase
– Vectors
•
•
•
•
Plasmid
Bacteriophage (lambda)
Cosmid
Yeast artificial chromosome
Screening
• Bacterial gene library
– Depends on protein expression
• Phenotype change e.g. cellulase gene
• Complementation of E. coli mutant
• Detection of gene product using antibody
• Eukaryotic gene library
– No protein expression expected
– Nucleic acid hybridisation
– Probe
• Homologous gene from other organism
• Oligonucleotide probe based on protein
sequence
Cloning cDNA
• cDNA: complementary or copy DNA
• It is derived from mRNA
• double stranded DNA with identical
sequence to mRNA in one strand
• Why clone cDNA
– Important in expressing eukaryotic proteins
in bacteria
– Introns not removed in bacteria
– Therefore not protein produced
Eukaryotic
organism
Bacteria
Transcription
RNA
Splicing
Translation
mRNA
Translation
Functional
protein
Inactive protein
Bacteria
cDNA
Transcription
mRNA
Translation
Functional
protein
Isolate specific cDNA
(encodes growth hormone)
• How do isolate mRNA encoding growth
hormone
• Isolate cells that express growth
hormone
– Pituitary
Isolate specific cDNA
(encodes growth hormone)
•
•
•
•
Extract mRNA
Convert to cDNA
Construct cDNA library
Screen library for members containing
cDNA encoding growth hormone
Pituitary
Isolate total mRNA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
Encodes GH
Purify mRNA
AAAAAA
AAAAAA
Matrix is
polydT
cellulose
TTTTTT
AAAAAA
TTTTTT
AAAAAA
Pass total RNA over
matrix. Only mRNA
will bind. Other RNAs
will pass directly
through
Ribosomal RNA etc
TTTTTT
AAAAAA
TTTTTT
AAAAAA
Low salt buffer
AAAAAA
TTTTTT
+
TTTTTT
AAAAAA
cDNA synthesis
• Use three enzymes
– reverse transcriptase
– RNAase H
– DNA polymerase
• Reverse transcriptase
– Converts RNA into a complementary DNA
sequence
– Add onto existing double stranded nucleic
acid
– Can’t initiate DNA synthesis
– Use primer to initiate DNA synthesis
primer
5’
3’mRNA5’
3’
Reverse
transcriptase
5’
3’
5’
3’
3’
5’
Primer
• Small single stranded DNA sequence
• Binds to 3’ end of all mRNA
• What is the sequence?
– PolydT
mRNA
5’
mRNA
5’
mRNA
cDNA
5’
3’
5’
3’
5’
3’
5’
3’
3’
AAAAAA
Add primer
TTTTT
3’
AAAAAA
TTTTTT
Reverse transcriptase
+ dNTPs
3’
AAAAAA
5’
TTTTTT
RNAase H
3’
AAAAAA
5’
TTTTTT
DNA polymerase + dNTPs
3’
AAAAAA
5’
TTTTTT
Completed DNA synthesis
3’
AAAAAA
5’
TTTTTT
Double stranded cDNA
Collection of cDNA molecules
• Insert cDNAs into a vector
– Generate recombinant vectors
• Insert recombinant vectors into a
bacterium such as Escherichia coli
• This comprises a cDNA library derived
from pituitary mRNA
• Screen cDNA library for those
containing growth hormone encoding
cDNA
cDNA is blunt-ended thus ligates
to other DNA unless concentration high
cDNA
GAATTC
CTTAAG
Add linkers at high concentrations
and DNA ligase
GAATTC
CTTAAG
GAATTC
CTTAAG
Cut with EcoRI
AATTC
G
G
CTTAAG
Insert into a vector
cut with same enzyme
followed by DNA ligase
Linkers
• Small complementary oligosaccharides
– Synthesised in large amounts
• Contain sequence cleaved with a restriction
enzyme
• Blunt ended double strand molecules
• Because synthesised at high concentrations
can be ligated efficiently to other double
stranded molecules (cDNA molecules)
• Once ligated to cDNA cut with restriction
enzyme to create sticky ends
cDNA is blunt-ended thus ligates
to other DNA unless concentration high
cDNA
GAATTC
CTTAAG
Add linkers at high concentrations
and DNA ligase
GAATTC
CTTAAG
GAATTC
CTTAAG
Cut with EcoRI
AATTC
G
G
CTTAAG
Insert into a vector
cut with same enzyme
followed by DNA ligase
Transform into
E. coli
Screen for members of
library that contain
growth hormone cDNA