Transcript Document

CHAPTER
3
Recombinant
DNA Technology
and Genomics
PowerPoint® Lecture by:
Melissa Rowland-Goldsmith
Chapman University
Chapter Contents
• 3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• 3.2 What Makes a Good Vector?
• 3.3 How Do You Identify and Clone a Gene
of Interest?
• 3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• 3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• 1970s: Gene cloning became a reality
– Clone – a molecule, cell, or organism that
was produced from another single entity
• Made possible by the discovery of:
– Restriction Enzymes – DNA cutting enzymes
(molecular scissors)
– Plasmid DNA Vectors – circular form of
self-replicating DNA
• Can be manipulated to carry and clone other
pieces of DNA
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Restriction Enzymes
– Primarily found in bacteria
– Cut DNA by cleaving the phosphodiester bond that
joins adjacent nucleotides in a DNA strand
– Bind to, recognize, and cut DNA within specific
sequences of bases called a restriction site
• Each restriction site is a palindrome – reads same forward
and backward on opposite strands of DNA
– There are 4 or 6 bp cutters because they recognize
restriction sites with a sequence of 4 or 6 nucleotides
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Why don't restriction enzymes digest bacteria DNA?
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Restriction enzymes
a. Some cut DNA to create DNA fragments with
overhanging single stranded ends called
"sticky" or "cohesive" ends
b. Some cut DNA to generate fragments with
double-stranded ends called "blunt" ends
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Would EcoRI cut the following sequence? Work in
groups to explain your answer.
• 5'CTCGAGTTCGAG3'
• 3'GAGCTCAAGCTC5'
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Restriction enzymes
• Advantage of enzymes that produce sticky ends
– Preferred for cloning because DNA fragments with
sticky ends can be easily joined together because
they base pair with each other by forming weak
hydrogen bonds
– Work in groups and use Table 3.1 to choose two
enzymes you would use for cloning and then state
your reasons.
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Plasmid DNA – small circular pieces of DNA
found primarily in bacteria
• Are considered extrachromosomal DNA because
they are in the cytoplasm in addition to the
bacteria chromosome
• Are small approximately 1 to 4 kb
• Can replicate independently of chromosome
• Can be used as vectors – pieces of DNA that
can accept, carry, and replicate other pieces of
DNA
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Creating recombinant DNA
• Look at Table 3.1, if you used the restriction enzyme
SmaI for both the insert and plasmid, would there be
hydrogen bonds of the blunt ends?
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Recombinant DNA
• 1975 NIH formed the RAC (Recombinant
DNA Advisory Committee) based on results
from Asilomar meeting
– Purpose of RAC: evaluate recomb. technology
and establish guidelines for research
– 1976 RAC published set of guidelines for
working with recomb. Organisms
• Work in groups to discuss one situation that could be
harmful to humans if RAC did not exist
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Transformation of Bacterial Cells
– very inefficient process
– A process for inserting foreign DNA into bacteria
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Treat bacterial cells with calcium chloride
Add plasmid DNA to cells chilled on ice
Heat the cell and DNA mixture
Plasmid DNA enters bacterial cells and is replicated and
express their genes
– electroporation
– Apply brief pulse of high voltage electricity to create
tiny holes in the bacteria cell wall that allow the DNA
to enter
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Selection of recombinant bacteria after
transformation
– Selection is a process designed to facilitate the
identification of recombinant bacteria while preventing
the growth of non-transformed bacteria and bacteria that
contain plasmid without foreign DNA
1. Antibiotic selection – plate transformed cells on plates
containing different antibiotics to identify recombinant
bacteria and non-transformed bacteria
– Does not select for plasmid containing foreign DNA vs.
recircularized plasmid
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Selection of recombinant bacteria after
transformation
2. Blue-white selection
– DNA is cloned into the restriction site in the lacZ gene
– When it is interrupted by an inserted gene, the lacZ
gene cannot produce functional Beta gal
– When Xgal (artificial lactose) is added to the plate, if
functional lacZ is present = blue colony
– Non-functional lacZ = white colony = clone =
genetically identical bacterial cells each containing
copies of recomb. plasmid
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
© 2013 Pearson Education, Inc.
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Assume you used a plasmid that contains the
lacz gene in the restriction enzyme site. The
plasmid has an antibiotic resistance gene.
Following transformation, you grow up the cells
on an agar plate containing the antibiotic. Here
are your results.
• Work in groups of two and discuss which
colonies have the inserted gene.
1
2
3
5
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4
6
3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• Introduction to human gene cloning
• First human protein expressed via recombinant
techniques was insulin and next was growth
hormone
• Clone human insulin DNA sequence into a plasmid
and the bacteria cells were then used to synthesize
the protein product of the cloned gene
• Can generate lots of pure protein via this technique
• What was source of growth hormone prior to
recombinant technology?
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Practical Features of DNA Cloning Vectors
– Size – small enough to be separated from chromosomal
DNA of host plasmid
– Origin of replication (ori) – site for DNA replication that
allow plasmids to replicate independently from host
chromosome
• Copy number: number of plasmids in the cell (normally small but
plasmids have high copy numbers)
– Multiple cloning site (MCS) – recognition sites for
several restriction enzymes in which insert is cloned into
– Selectable marker genes – allow to select for
transformed colonies
– RNA polymerase promoter sequences – used for
transcription in vitro and in vivo
– DNA sequencing primers
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Types of Vectors
– Bacterial plasmid vectors – can clone inserts
that are smaller than 7 kb; some express
eukaryotic proteins from genes poorly
– Bacteriophage vectors
– Cosmid vectors
– Expression vectors
– Bacterial Artificial Chromosomes (BAC)
– Yeast Artificial Chromosomes (YAC)
– Ti vectors
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Types of vectors
– Bacteriophage vectors – advantage: clone up to 25
kb λ genome is linear and 49 kb
– Cloned DNA is inserted into restriction sites in center
of λ chromosome
– Recomb. chromosomes are packaged into viral
particles in vitro
– These phages then infect lawn of E coli cells
– At each end of λ are 12 bp sites = COS which base
pair together when they infect bacteria and circularize
and replicate
– Obtain plaques – zones of dead bacteria which
contain millions of recombinant phage particles
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Cosmid vectors: contain
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•
•
•
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COS ends of λ DNA
Plasmid ori of rep
Gene for antibiotic resistance
DNA is cloned into restriction site
Then cosmid is packaged into viral particles and
used to infect E coli cells at low copy number
• Bacterial colonies are grown on the plate and
recombinants are screened by antibiotic selection
Advantages: clone fragments between 20–40 kb
• Discuss the cosmid features shared by either
a plasmid and bacteriophage
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Expression vectors
– Bacteria expression vector:
• Allow high level protein expression in bacterial cells because
they have a prokaryotic promoter site next to the MCS
• Bacterial RNA POL can then bind to promoter and transcribe
the insert's sequence which is then translated into protein
• Protein is then purified using biochem. techniques
• Problems with bacteria expression vectors: sometimes
bacteria ribosomes cannot translate eukaryote sequence or
protein is not folded correctly since bacteria does not have
organelles for processing. It is not possible to use this system
with eukaryote genomic DNA.
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Assume you want to make lots of human
insulin using a bacteria expression
vector.
• Work in groups of two and discuss why
using human insulin genomic DNA for
this cloning project would not be
advantageous.
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Bacteria artificial chromosomes (BAC)
– Large low copy plasmids
– Contain genes that encode the F factor (unit
of genes controlling bacterial replication)
– Can accept large sizes of DNA inserts ranging
from 100–300 kb
– Were used during the human genome project
to clone and sequence large pieces of
chromosomes
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Yeast artificial chromosomes (YAC)
– Is miniature version of eukaryotic
chromosome containing ori or rep; two
telomeres; selectable markers; centromere
that allows replication of YAC and segregation
of daughter cells
– Best for cloning very large DNA inserts from
200 kb to 2 megabases
– Were used for human genome project
– Small plasmids grown in E coli and introduced
to yeast cells (S. cervisiae)
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
• Ti vector
– Naturally occurring plasmids isolated from the
bacterium that is a soil plant pathogen
causing disease in plants
– When the bacteria infects plant cells, the T
DNA from the Ti plasmid inserts into the host
chromosome
– T DNA codes for auxin hormone that weakens
plant cell wall and infected plants divided and
enlarge to form a tumor (gall)
– Scientists use Ti vectors to deliver genes to
plants by removing toxic gene for auxin
© 2013 Pearson Education, Inc.
3.2 What Makes a Good Vector?
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Creating DNA Libraries
– Collections of cloned DNA fragments from a
particular organism contained within bacteria or
viruses as the host
– Screened to pick out different genes of interest
• Two Types of Libraries
– Genomic DNA libraries
– Complementary DNA libraries (cDNA
libraries)
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Genomic Libraries
– Chromosomal DNA from the tissue of interest
is isolated and digested with a restriction
enzyme which produces many fragments that
include the entire genome
– Vector is digested with same enzyme
– DNA ligase is used to ligate genomic DNA
fragments and vector DNA
– Recombinant vectors are used to transform
bacteria and theoretically each bacteria will
contain a recombinant plasmid
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
– Disadvantages of genomic libraries
• Introns are cloned in addition to exons;
– Majority of genomic DNA is introns in eukaryotes so
majority of the library will contain non-coding pieces of
DNA
• Many organisms have very large genome, so
searching for gene of interest is difficult
• Time consuming!
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• cDNA Libraries
– mRNA from tissue of interest is isolated
– Need to make double stranded DNA from mRNA: How?
a. enzyme reverse transcriptase catalyzes synthesis of
complementary single stranded DNA from mRNA
i. Called complementary DNA (cDNA) because it is an exact
copy of the mRNA
b. mRNA is degraded either with an enzyme or alkaline solution
c. DNA Pol is used to synthesize second strand of DNA to create
double stranded cDNA
– Short linker double stranded DNA sequences which contain
restriction enzyme recognition sites are added to the ends of the
cDNA
– Cut with restriction enzyme, cut vector with same enzyme, ligate
fragments to create recombinant vectors
– Then transform bacteria with recombinant vectors
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• cDNA Libraries
– Advantage over genomic libraries
• Collection of actively expressed genes in the cells or tissues from
which the mRNA was isolated
• Introns are NOT cloned
• Can be created and screened to isolate genes that are primarily
expressed only under certain conditions in a tissue
• Assume that a gene involved in increased muscle mass is
expressed when the muscle cells are exposed to growth hormone.
What would be the source of the cDNA library: muscle cells or
muscle exposed to growth hormone? Work in groups to explain
your answer.
– Disadvantage
• Can be difficult to make the cDNA library if a source tissue with
an abundant amount of mRNA for the gene is not available
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Library screening to identify the gene of interest
• Colony hybridization
– Bacterial colonies containing recombinant DNA are grown on an
agar plate
– Nylon or nitrocellulose filter is placed over the plate and some of
the bacterial colonies stick to the filter at the exact location they
were on the plate
– Treat filter with alkaline solution to lyse the cells and denature
the DNA
– Denatured DNA binds to filter as single-stranded DNA
– Filter is incubated with a probe that is tagged with a radioactive
nucleotide or fluorescent dye
• DNA fragment that is complementary to the gene of interest
– Probe binds by hydrogen bonding to complementary sequences
on the filter = hybridization
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Why is it necessary to tag the probe
with either a radioactive nucleotide or
fluorescent dye that can be used to
catalyze light-releasing reactions?
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Colony Hybridization
– Filter is washed to remove excess unbound probe
– Filter is exposed to film – autoradiography
• Anywhere probe has bound to the filter, radioactivity from the
radioactive probe or released light (fluorescence or
chemiluminescence) from non-radioactive probes exposes silver
grains in the film
– Depending on the abundance of the gene of interest there might be
few colonies or plaques on the filter that hybridize to the probe
• Film is developed to create a permanent record of the colony
hybridization
– Use digital instrument to detect probe binding if a fluorescent or
chemiluminescent probe was used
– Film is then compared to the original agar plate to
identify which colonies contained recombinant plasmid
with the gene of interest
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Colony Hybridization
– Type of probe used depends on what is already known about the
gene of interest
– Example: use mouse or rat probe to screen a human library
because many genes between these species are similar
– If gene sequence has NOT been cloned in another species but
something is known about the protein, what can be done?
• Library screening rarely results in the cloning of the fulllength gene
– Usually get small pieces of the gene; the pieces are sequenced
and scientists look for overlapping sequences
– Look for start and stop codons to know when the full length of the
gene is obtained
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Polymerase Chain Reaction
– Developed in the mid-1980s by Kary Mullis
– Technique for making copies, or amplifying, a specific
sequence of DNA in a short period of time
– Process
• Target DNA to be amplified is added to a tube, mixed with
nucleotides (dATP, dCTP, dGTP, dTTP), buffer, and DNA
polymerase.
• Paired set of Forward and Reverse Primers are added –
short single-stranded DNA oligonucleotides (20–30bp long)
– Primers are complementary to nucleotides flanking opposite
ends of target DNA
• Reaction tube is placed in an instrument called a
thermocycler
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• PCR Process continued
– Thermocycler will take DNA through a series of
reactions called a PCR cycle
– Each cycle consists of three stages
1. Denaturation – heat to 94 °C to 96 °C
2. Annealing (hybridization) – in which primers H bond
with complementary bases at the opposite ends of
target sequence at 55 °C to 65 °C
3. Extension (elongation) – DNA Pol copies target DNA at
70 to 75 °C
– At the end of one cycle, the amount of DNA has
doubled
– Cycles are repeated 20–30 times
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Advantage of PCR:
• Can amplify millions of copies of target DNA from
small amount of starting material in short period
of time
• To calculate the number of copies of target DNA
starting with 1 molecule of DNA use this equation
2N in which N represents number of PCR cycles
• Assume you want to do 22 PCR cycles to
amplify your DNA insert, how many copies of
DNA will you have at the end of your PCR?
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• The type of DNA polymerase used is very important
– Taq DNA polymerase – isolated from a species known as
Thermus aquaticus that thrives in hot springs
– Why can't you use DNA Pol isolated from bacteria
that live at 37 C?
• Applications
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Making DNA probes
Studying gene expression
Detection of viral and bacterial infections
Diagnosis of genetic conditions
Detection of trace amounts of DNA from tissue found at
crime scene
– Detection of DNA from fossilized dinosaur tissue
© 2013 Pearson Education, Inc.
How Do You Identify and Clone a Gene of
Interest?
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
• Cloning PCR Products
– Is rapid and effective compared to using DNA libraries
– Disadvantage
• Need to know something about the DNA sequence that
flanks the gene of interest to design primers
• Assume the human genome project was not completed
but you wanted to clone growth hormone from humans,
what sequence would you use to design PCR primers?
– Great trick: Taq polymerase puts a single adenine
nucleotide on the 3' end of all PCR products
• Use this knowledge to researcher's advantage: T vector that
has single stranded thymine on each end so can
complementary base pair with the adenine in the PCR
products
© 2013 Pearson Education, Inc.
3.3 How Do You Identify and Clone a Gene
of Interest?
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Agarose Gel Electrophoresis: separate and visualize DNA fragments
based on size
• Agarose is isolated from seaweed, melted in a buffer solution and
poured into a horizontal tray. As it cools, it will form a semisolid gel
containing small pores through which DNA will travel
• The percentage of agarose used to make the gel determines the
ability of the gel to separate DNA fragments of different sizes
• (gel % range from 0.5 to 2%)
• High % gel (2%) allows to resolve smaller size fragments
• Low % (0.5%) resolves larger size fragments
• What size fragments would be resolved using a 1% gel?
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Agarose Gel Electrophoresis
– To run a gel, it is submerged in a buffer solution that
conducts electricity
– DNA is loaded into small depressions called wells at
the top of the gel
– Electric current is applied through electrodes at
opposite ends of the gel
• DNA migrates according to its charge and size
• Rate of migration through the gel depends on the size of the
DNA because the sugar phosphate backbone makes it
always negatively charged
• DNA migrates toward positive pole and is repelled by
negative pole
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Agarose Gel Electrophoresis
• Migration distance is inversely proportional to size
of DNA fragment
– Large fragments migrate slowly; smaller fragments
migrate faster
– Tracking dye is added to the samples to monitor DNA
migration during electrophoresis
– DNA can be visualized after electrophoresis by the
addition of DNA staining dyes
• Ethidium bromide: intercalate between DNA base pairs and it
fluoresces under ultraviolet light
• Then a picture can be taken to document the gel results
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
M Digest
basepairs
2000
1500
1000
A
B
C
500
• Here is a result in which you ran your DNA fragments on a 1%
gel. What is the approximate size of band B? Why did it not
migrate super fast through the gel?
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Restriction mapping gene structure
– Cut cloned gene with restriction enzymes to pinpoint
location of the cutting sites
– Knowing restriction map is useful for making clones of
small pieces of the DNA which is called subcloning
– These small pieces of DNA can then be sequenced
– Protocol of restriction mapping:
a. Digest DNA with single or double restriction enzymes
b. Separate DNA fragments via agarose gel electrophoresis
c. Arrange fragments in order to make map of restriction
sites
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
*** Note: These days scientists use bioinformatic software
to identify restriction sites in the DNA
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• DNA Sequencing
– Important to determine the sequence of nucleotides of
the cloned gene
– Work in groups of two to discuss at least three
reasons why it is important to know the sequence of
the cloned DNA
– Chain termination sequencing (Sanger method)
– Requires single stranded primer annealing to denatured
DNA template. The reaction tube also contains all 4
dNTPs, DNA Pol, and dideoxynucleotide (ddNTP)-which has a 3' H instead of 3'OH on the deoxyribose. It
cannot form a phosphodiester bond with the incoming
nucleotide and so gets terminated.
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Original Sanger method:
– Had four separate reaction tubes and each contained the vector;
primer; dNTPs in which one dNTP is radioactively labeled; and a
different small amount of ddNTP; DNA Pol.
– Over time, a ddNTP will be incorporated into all the positions in the
newly synthesized strands creating fragments of varying lengths
that are terminated at the ddNTP.
– Then the fragments are separated on polyacryamide gel.
– Autoradiography used to then identify radioactive fragments.
– Read the gel from bottom to top as individual nucleotides.
– The sequence generated from the reaction is complimentary to the
sequence on the template strand in the vector.
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Read the first 24 nucleotide sequence from the gel
generated in Figure 3.12
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
– High throughput computer automated sequencing using
Sanger method--instead of sequencing 200–400
nucleotides per reaction, this allowed greater than 500
nucleotides to be sequenced per reaction.
– Was very helpful for completing human genome project
– Procedure: instead of 4 separate tubes, only 1 reaction
tube is used
• ddNTPs are each labeled with a different fluorescent dye
• Samples are separated on a single-lane capillary gel that is
scanned with a laser beam
• Laser stimulates fluorescent dye on each DNA fragment which
emits a different wavelength of light for each different colored
ddNTP
• Emitted light is collected by a detector that amplifies and feeds
this info. to a computer that can run multiple capillary gels at one
time = 900 bp sequence
• Computer converts light patterns to reveal the sequence
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
•
•
•
•
Next generation sequencing (NGS) = designed to produce highly accurate and
long stretches of DNA sequence (greater than 1 gigabase) of DNA per reaction
Is done at very low cost
Use parallel formats that use fluorescence imaging techniques
Two types of NGS approaches
A. Roche 454 commercial system using pyrosequencing:
– Pyrosequencing: beads are attached to fragmented genomic DNA which is
PCR amplified in separate water droplets in oil for each bead then loaded
into multiwell plates and mixed with DNA POL; then actual pyrosequencing
– Pyrosequencing: single dNTP is flowed over the wells and if it incorporated
into complementary sequencing strand, a pyrophosphate is released due to
making a phopshodiester bond and then a series of chemiluminescent (light
releasing) reactions take place to ultimately produce light with firefly
luciferase enzyme
• Emitted light from the reaction is captured and recorded to determine
when a single nucleotide has been added to the growing strand
• By rapidly repeating the nucleotide flow step with each of the 4 dNTPs,
can obtain reads of approx. 400 nucleotides and order of 400 million
bases of data per 10 hour run
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• The other type of NGS approach:
B. ABI SOLID (supported oligonucleotide ligation and detection)
method
– Can produce 6 gigabases of sequence data per run
– Similar to 454 approach in which DNA fragments are linked to
beads and amplified but different sequencing technologies used
C. Future = third generation sequencing using nanotechnology
to push single strand nucleotide fragments into nanopores
and then cleaving off individual bases to produce a signal
that can be captured
– This new approach will not involve DNA amplification or fluorescent tags
so it is truly direct sequencing
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Fluorescence in situ hybridization (FISH)
• Chromosome Location and Copy Number
– Identify which chromosome contains a gene of interest
– Procedure:
• Chromosomes are isolated from cells and spread out on glass
microscope slide
• DNA or RNA probe for gene of interest is labeled with
fluorescent nucleotides and incubated with slides
• Probe will hybridize with complementary sequences on
chromosomes on slide
• Slide is washed and exposed to fluorescent light
• Wherever probe has bound to the chromosome, it is illuminated
to indicate the presence of the probe binding
• Do karyotype to determine which chromosome shows
fluorescence
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
– Fluorescence in situ
hybridization (FISH) continued
• To determine which chromosome
shows fluorescence they are
aligned according to their length
and staining patterns of their
chromatids to create a karyotype
• What does fluorescence on more
than one chromosome indicate?
• FISH used to analyze genetic
disorders
• FISH used to determine which cells
in a particular organ are expressing
the particular mRNA
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
– Southern blotting
– Used to determine gene copy number; gene
mapping; gene mutation detection; PCR product
confirmation; DNA fingerprinting
• Digest chromosomal DNA into small fragments with
restriction enzymes
• Fragments are separated by agarose gel electrophoresis
• Gel is treated with alkaline solution to denature the DNA
• Fragments are transferred onto a nylon or nitrocellulose filter
(called blotting)
• Filter (blot) is baked or exposed to UV light to permanently
attach the DNA
• Filter (blot) is incubated with a labeled probe and exposed to
film by autoradiography
• Number of bands on film represents gene copy number
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
– Techniques involve analyzing mRNA produced
by a tissue
– Northern blot analysis
• Basic method is similar to Southern blotting
• RNA is isolated from a tissue of interest, separated
by gel electrophoresis, blotted onto a membrane,
and hybridized to a labeled DNA probe, exposed
bands on autoradiograph show presence of mRNA
for gene of interest as well as size of mRNA
• Can compare and quantify amounts of mRNA
present in different tissues
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene
Expression
– Techniques involve
analyzing mRNA
produced by a tissue
– Northern blot analysis
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
• Reverse transcription PCR- used to study mRNA levels when level of
detection is below that of Northern
• Procedure:
• Isolate mRNA and use Reverse Transcriptase to make double
stranded cDNA
• What procedure did you already learn about using RT in this
chapter?
• Use PCR to amplify region of cDNA with set of primers specific for
gene of interest
• Run agarose gel to separate amplified fragments
• Determine expression patterns in the tissue
• **Amount of cDNA produced in RT PCR rxn for gene of interest
reflects amount of mRNA and level of gene expression
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
• Real time or quantitative (qPCR)
• Can quantify amplification reactions as they occur in real time
• Need special thermal cyclers that use a laser to scan a beam
of light through the top or bottom of each PCR reaction
• Each reaction tube contains EITHER a dye containing probe
or DNA binding dye that emits fluorescent light when
illuminated by the laser
• Light emitted by the dyes correlates with amount of PCR
product amplified
• Light is captured by the detector which relays info. to the
computer to provide readout on amount of fluorescence
• Readout is plotted and analyzed to quantitate the number of
PCR products produced after each cycle
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
•
•
Real time or quantitative (qPCR)
Two approaches
a. Taqman probes are complimentary to specific
regions of target DNA between forward and reverse
primers for PCR
– Taqman probes contain two dyes: reporter located at 5' end
of probe and can release fluorescent light when excited by
the laser and other dye is quencher which is attached to 3'
end of probe
(see figure for more details)
B. SYBR green- binds double stranded DNA and as
more double stranded DNA is copied with each round
of qPCR there are more DNA copies to bind SYBR
Green which increases amount of fluorescent light
emitted
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
– Gene microarrays enable researchers to
study all of the genes expressed in a tissue
very fast
– Microarray (gene chip) is created with use of
small glass microscope slide
• Single stranded DNA molecules are spotted on the
slide using an arrayer (computer controlled robotic
arm) which fixes DNA (multiple copies of cDNA) at
different spots on the slide which is recorded by a
computer
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
– Gene microarrays
• Procedure to do array:
– Extract mRNA from a tissue of interest and cDNA is
synthesized from mRNA and labeled with fluorescent dye
– Labeled cDNA is incubated overnight with the array
where it hybridizes with different spot on the array that
contain complementary DNA sequences
– Can have over 10,000 spots of DNA
– Array is washed and scanned by a laser that causes
cDNA hybridized to array to fluoresce
– Fluorescent spots reveal which genes were regulated
and Intensity of fluorescence indicates relative amount of
gene expression
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
• Studying Gene Expression
– Gene microarrays
– Common to do microarray in which you compare two
different conditions with 2 different colored dyes (one
for treatment and other for control condition)
– Laser is scanned at different wavelengths for each
probe and then the images are overlaid to make
direct comparisons between the treatment and control
• Example study gene expression difference between cancer
cells and normal cells to look for genes possibly involved in
cancer progression
• Results of such studies can possibly lead to new drug
therapies to combat cancer and other diseases
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
– Gene mutagenesis studies
• To study structure and function of protein produced by a
specific gene
• Site directed mutagenesis: mutations created in specific
nucleotides of a cloned gene contained in a vector
• Gene is then expressed in cells which results in translation of
a mutated protein
• This procedure lets researchers study effects of mutation on
protein structure as well as provides information about which
nucleotides are important for specific functions of a protein
• Very useful in helping identify critical sequences in a gene
that produced proteins involved in disease
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
– RNA interference (RNAi) = naturally occurring
mechanism for inhibiting gene expression
Procedure:
– Doublestranded RNA can be bound by Dicer enzyme
that cuts dsRNA into 21–25 nucleotide snippets called
small interfering RNA (siRNA)
– siRNA then bound to protein: RNA complex called RNA
inducins silencing complex (RISC)
– RISC unwinds dsRNA releasing single stranded RNA
that bind complementary mRNA
– Binding of siRNA to mRNA leads to degradation of
mRNA by Slicer enzyme or blocks translation by
interfering with ribosome binding
– Currently developing techniques to use RNAi to silence
gene expression
© 2013 Pearson Education, Inc.
3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• Genomics – cloning, sequencing, and
analyzing entire genomes
– Whole genome shotgun sequencing or shotgun
cloning
• Use restriction enzymes to digest pieces of entire
chromosomes:
– Produces thousands of overlapping fragments called
contigs which are each sequenced.
– Computer programs are used to align the sequenced
fragments based on overlapping sequence pieces
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• Bioinformatics: Merging molecular biology with
computer technology
– An interdisciplinary field that applies computer
science and information technology to promote an
understanding of biological processes
• Application of Bioinformatics
– Databases to store, share, and obtain the maximum
amount of information related to gene structure, gene
sequence and expression, and protein structure and
function
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
– GenBank = public database of DNA sequences
and contains National Institute of Health
collection of DNA sequences
• Each entry has an accession number that scientists
use to refer back to the cloned sequence
• Maintained by the National Center for Biotechnology
Information (NCBI)
– NCBI is goldmine for bioinformatics resources that creates
public access databases and develops computing tools for
analyzing and sharing genome data
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
– DNA database searching
• Basic Local Alignment Search Tool (BLAST)
– Used to search GenBank for sequence matches between
cloned genes and to create DNA sequence alignments
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• The Human Genome Project
– Started in 1990 by the U.S. Department of
Energy
– International collaborative effort to identify all
human genes and to sequence all the base
pairs of the 24 human chromosomes
– Mostly done by 20 centers in 18 countries:
China, France, Germany, Great Britain, Japan,
and the United States
– Competitor was a private company, Celera
Genomics, directed by Dr. J. Craig Venter
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• The Human Genome Project designed to accomplish the
following:
• Analyze genetic variations among humans. This included the
identification of single-nucleotide polymorphisms
• Map and sequence the genomes of model organisms, including
bacteria, yeast, roundworms, fruit flies, mice, and others
• Develop new laboratory technologies such as high-powered
automated sequencers and computing technologies, as well as
widely available databases of genome information, which can be
used to advance our analysis and understanding of gene structure
and function
• Disseminate genome information among scientists and the general
public
• Consider the ethical, legal, and social issues that accompany the
HGP and genetic research
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• The Human Genome Project
– April 14, 2003: map of the human genome was
completed
– Consists of 20,000 protein-coding genes
– Map was complete with virtually all bases identified
and placed in their order and potential genes
assigned to chromosomes
• Why are there only 20,000 genes coding for proteins
when it was predicted that there would be 100,000
genes? Work in pairs to answer this question.
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• What we have learned from HGP
– Nearly 50% genes do not yet have a function
• Summary of findings:
– The human genome consists of approximately 3.1 billion base
pairs
– The genome is approximately 99.9% the same between individuals
of all nationalities
– Single-nucleotide polymorphisms (SNPs) and copy number
variations (CNVs)—such as long deletions, insertions and
duplications in the genome—account for much of the genome
diversity identified between humans
– Less than 2% of the genome codes for genes
– The vast majority of our DNA is non-protein coding, and repetitive
DNA sequences account for at least 50% of the noncoding DNA
– The genome contains approximately 20,000 protein-coding genes
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
•
•
•
•
•
What we have learned from HGP
Many human genes are capable of
making more than one protein,
allowing human cells to make at
least 100,000 proteins from only
about 20,000 genes.
Chromosome 1 contains the
highest number of genes. The Y
chromosome contains the fewest
genes.
Many of the genes in the human
genome show a high degree of
sequence similarity to genes in
other organisms.
Thousands of human disease
genes have been identified and
mapped to their chromosomal
locations
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• The Human Genome Project
– Started an "omics" revolution
• Proteomics—studying all proteins in a cell
• Metabolomics—studying proteins and enzymatic
pathways involved in cell metabolism
• Glycomics—studying carbohydrates of a cell
• Transcriptomics—studying all genes transcribed in a
cell
• Metagenomics—analysis of genomes of organisms
in an environment
• Pharmacogenomics—customized medicine based
on person's genetic profile for a particular condition
• Nutrigenomics—interaction between genes and diet
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• Comparative Genomics
– Mapping and sequencing genomes from a
number of model organisms
– Allows researchers to study gene structure
and function in these organisms in ways
designed to understand gene structure and
function in other species including humans
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• Stone Age Genomics (paleogenomics)
– Analyzing "ancient" DNA
© 2013 Pearson Education, Inc.
3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
• 10 years after completion of HGP—what next?
– Human epigenome project—create maps of epigenetic
changes in different cell and tissue types
– International Hapmap Project—characterization of
SNPs for their role in genome variation, disease and
pharacogenomics applications
– Encyclopedia of DNA elements (ENCODE)—use
experimental approaches and bioinformatics to ID and
analyze functional elements that regulate expression of
human genes
– Personalized genome projects
– Cancer genome project—map important genes and
genetic changes involved in cancer
© 2013 Pearson Education, Inc.