MAKING A PROBE :

- Preference is for nucleic acid probes rather than

proteins. Why?

- Properties: Unique, not too short or too long

- How to make a probe?

METHODS FOR MAKING A PROBE:

A. Cellular Extraction of mRNA then convert to cDNA

- If mRNA is known
- Extract the target mRNA and by binding to a
Poly T cellulose column
then elute the bound mRNA using buffer with
low salts then generate cDNA using a specific
gene as primer (random primer method)

- If mRNA is not known

- Extract all mRNA from cell and use oligo-dT
primer rather than a gene primer
METHODS FOR MAKING A PROBE:

A. Assume probe for beta globin gene, mRNA

available

- Extract total RNA from reticulocytes

- Purify m-RNA from mixture by running on

oligo-dT-cellulose column chromatography
Figure 2.11
METHODS FOR MAKING A PROBE:

B. Chemical Synthesis of DNA (p. 42-48 Ch 2):

Link one nucleotide at a time: link nucleotide to

solid support, activate the P at which

DNA will be added by a methylated 3 phosphite

group, Block reactive groups by DMT,

Purify by HPLC
Chemical Synthesis of DNA

The phosphoramadite method is the most commonly

used method of synthesis of oligonucleotides

It is a multistep process of detritylation,

phosphoramadite activation, coupling, capping and
oxidation cycles

Addition of nucleotides is opposite to biological

process, it happens in 5’ to 3’ direction
Figure 2.25

Column washed
with acetonitrile
to remove water

Detritylation
involves
treatment with
tricholoracetic
acid (TCA) to
activate the 5’-
OH group

Washing with
acetonitrile to
remove TCA and
then with argon
to remove
acetonitrile
Figure 2.26

Binding of the first nucleoside

to CPG beads (inert support)
Figure 2.27
Figure 2.28

CPG: controlled pore glass bead which is bead of glass with uniform size pores
Figure 2.29
 Not all support bound nucleosides are linked to phosphoramadite
during first coupling reaction, thus unlinked residues should be
prevented from linking to next nucleotide. The unreacted 5’OH
Figure 2.30 group is thus acetylated by capping so that growing chains are of
same length
 Linkage between nucleotides is in form of phosphite triester
bond which is unstable, thus need to oxidize by iodine mixture to
form the more stable pentavalent phosphate triester
Figure 2.31
Table 2.3

To achieve reasonable yield, the coupling efficiency should be more than

98% at each step, most machines provide 99.5%
Coupling efficiency can be monitored by a spectrophotometer by
measuring absorbance of the released trityl groups
Better to purify DNA by reverse phase HPLC or gel electrophoresis to
remove shorter failure sequences
Assembling Oligos into Genes

• Possible to synthesize genes for large scale production of

proteins or for testing protein function upon change in
codons, or generating novel proteins by changing gene

• Less than 100bp is easy: make two complementary oligos

then anneal them

• If larger (1000bp) use tricks (p. 48 and 49 of textbook)

METHODS FOR MAKING A PROBE:

C. Nick translation:
Nick provides the free 3'OH end used by DNA
polymerase (2 Types: - Eukaryotic: only DNA
synthesis
- Prokaryotic: 3 activities
a) DNA synthesis
b) 3'5' exonuclease
c) 5'3' exonuclease

Add nick DNA + Klenow DNA polymerase+

4 dNTPs (*)
GENE SYNTHESIS BY PCR:
POLYMERASE CHAIN REACTION:

The assembly of a gene by PCR is faster than filling in

overlapping oligos using DNA polymerase

- Magnify small pieces of DNA

- Use Taq I DNA polymerase (from

thermophilic bacteria Thermophilus
aquaticus)

- 10 cycles  1000 fold magnification

Figure 2.33

Start
Figure 2.33 (Continued)
Q: How do scientists determine what primer sequences and
temperature conditions should be used for a PCR
experiment?
 MAKING RNA PROBES:

• Advantage of RNA probes?

• Phage SP6 system could be used to prepare labeled

RNA of high specific activity

 Detection by Autoradiography or Chemiluminescence
depending on type of Probe

After Preparing Probe Use Detection Methods

• Detect radioactivity in bands (DNA, RNA or protein)

• Use a photographic emulsion film sensitive for

radiation (made of silver bromide AgBr)

• Ag deposits when hit w/ radiation (dark bands)

• Place film above membrane following hybridization

• Incubate O.N. in freezer then develop in machine

APPLICATION OF CLONING, PROBING & HYBRIDIZATION
TECHNIQUES:

- VIROID detection in plants

- Allele specific probes to detect diseases based on
gene mutations (CFTR)
- DNA fingerprinting (used in court for crimes)
- Restriction Fragment Length Polymorphisms
- Cloning PCR products
Figure 4.24

DNA Fingerprints
from a Murder Case
DNA bloodstains on
the defendant’s shirt
match the DNA
fingerprints of the
victim but not the
defendant. This
indicates that the
victim’s blood got on
the defendant’s
clothes, placing the
defendant at the scene
of the crime.
Cloning PCR Products
TWO METHODS: with or without RE

• Amplify DNA by PCR then clone into

vector
• Method 1: Add RE recognition sites to
the 5’end of the primers
• Although these sites may not be
complementary to the target sequence,
they do not interfere with DNA synthesis
• After several cycles, DNA is magnified
and flanked by RE recognition sites
which could be cut with the RE to
generate sticky ends for ligation

P1: primer 1, P2: primer 2

RE recognition site in green
 Figure 2.22
Cloning PCR Products

• Method 2: Do not use RE sites

• Use Taq DNA polymerase to add a
single dAMP to the ends of amplified
DNA molecules
• Construct a cloning vector with dTMP
overhangs
• dAMP can base pair with
complementary dTMP
• Ligate with T4DNA ligase and insert
PCR product into vector without need to
cut with RE
Quantitative Real Time PCR
-PCR in which DNA or RNA
molecules are quantified in real
time
-Amount of DNA is measured by
measuring the amount of SYBR
green fluorescence, a dye that
gets incorporated only in double
stranded DNA
-Fluorescence intensity is
proportional to the concentration
of DNA
- Useful to quantify microbial
contamination in food samples
The Four Phases of Real Time PCR
• Linear Phase (10-15 cycles): fluorescence is at
background level and DNA cannot be quantified
• Early Exponential Phase: Fluorescence increases
above threshold and could be quantified (ie threshold
cycle-CT).
• Third Phase: Amount of fluorescence continues to
double as DNA products double in the cycle
• Fourth Plateau Phase: reaction components become
limited and not too useful to measure intensity of
fluorescence
Quantifying PCR product
Figure 2.24

Amount of DNA is quantified by using standards in which there is

a known number of copies of the target DNA
 Restriction Fragment Length
Polymorphisms

Detection of Traits in Populations

(e.g. Sickle cell anemia, thalassemia)
 DNA SEQUENCING: Why sequence DNA?
• Reveal function of the protein encoded by the gene (e.g
reveal DNA binding regions or receptor recognition sites)
• Nucleotide sequences in non coding regions can provide
information about the regulation of the gene
• Compare gene sequences between normal and unhealthy
individuals- know mutations that cause different phenotypes
• Polymorphisms (nucleotide differences) can help predict
susceptibility to diseases
 DNA Sequencing Projects and Steps
• denovo genome sequencing: genome of organism that has
not been sequenced before

• resequencing: comparing new sequence with a reference

sequence

Steps:
1. Prepare a library of template DNA fragments
2. Amplify the DNA fragments
3. Sequence the template DNA
4. Assemble the sequences generated from the fragments in
the order in which they are found in the original genome
c-DNA and Genomic Libraries
A. GENOMIC LIBRARY:
- The 1st library was of the beta-globin gene
- Human genomic DNA from reticulocytes
- Cut DNA with RE
- If 3 x 109bp, RE cuts at 400 bp  get 750,000
fragments  Screen many colonies
A. GENOMIC LIBRARY:

- Use partial digestion: RE concentration

Incubation time
- Phage lambda or cosmids are vectors of choice
- Colony hybridization is method to screen for
colonies that contain the gene of interest
B. c-DNA LIBRARY:
- Start with m-RNA comprising total cell mRNA
- Make a c-DNA of mRNA
- Synthesize second strand (complementary)
- Insert into vectors
- Culture and select colonies containing c-DNA
DNA SEQUENCING METHODS:
1. Maxam Gilbert Chemical Cleavage Method
2. Sanger Enzymatic Method based on M13
3. Sanger Method based on PCR-based cycle sequencing:
ddNTP procedure
4. Next Generation Sequencing (NGS):
• Pyrosequencing: based on detection of pyrophosphate during DNA
synthesis
• Sequencing using Reversible Chain Terminators (read p. 57)
• Sequencing by Single Molecule Synthesis (read p.57-58)
• Sequencing Whole Genomes by shotgun sequencing (read p. 59-60)

5. High Throughput NGS: replaced shotgun as there is no need

for cloning (cell free method). Attach, amplify, sequence
genomic DNA fragments directly on solid support (read p. 61-62)
 DNA SEQUENCING:

CLASSICAL METHODS:
B. Maxam & Gilbert (Chemical cleavage method)
- label 5' end of DNA w/ 32P
- Separate the two strands
- Divide DNA into 4 samples and treat each w/
chemical that cuts one or two of the 4 bases
- Generate a series of fragments, run on gel,
read sequence of analyzed strand
DNA SEQUENCING:
CLASSICAL METHOD:
A. Frederick Sanger method (Enzymatic):
- Sanger and Gilbert received Nobel
prize in 1980
- Phage M13 but later replaced by PCR-
based cycle sequencing
- Dideoxynucleoside triphosphates: once
incorporated into the DNA synth. stops
- Read the complementary synthesized
strand, deduce the gene sequence
Figure 2.34
Putting all four deoxynucleotides into
the picture:

Well, OK, it's not so easy reading

just C's, as you perhaps saw in the
last figure. The spacing between the
bands isn't all that easy to figure out.
Imagine, though, that we ran the
reaction with *all four* of the dideoxy
nucleotides (A, G, C and T) present,
and with *different* fluorescent
colors on each. NOW look at the gel
we'd get (at left). The sequence of
the DNA is rather obvious if you
know the color codes ... just read the
colors from bottom to top:
TGCGTCCA-(etc).
 Figure 2.35

• DNA molecules of
different sizes are
separated by capillary
electrophoresis and as
each molecule passes by
a laser signal is recorded

• We don’t even need to

read the sequence from
the gel, the computer
does that for us!

• Computer interprets the

colors by printing the
nucleotide sequence
across the top of the plot
 Principles of Pyrosequencing
• Pyrophosphate is
released during DNA
synthesis by cleavage of
Figure 2.36 bond between β and α
phosphates
• Add an adaptor
sequence that acts as a
primer is added to the 3’
end of DNA template
• One dNTP is added at a
time thus releasing
pyrophosphate
• PPi is detected by
synthesis of ATP
• When ATP is made, light
is generated by
luciferase
Shotgun Sequencing:

• Instrumental in the Human Genome Project

• Using Bacterial Artifical Chromosome (BAC), scientists
have cloned (replicated) major chunks of human DNA
which was critical to the Human Genome Project.
• Construct BACs that carry DNA from humans or mice,
and insert the BAC into a host bacterium.
• Huge pieces of DNA can be easily replicated using BACs -
usually on the order of 100-400 kbs
Construction and Sequencing of Metagenomic Libraries

Figure 2.43
Main purpose it to
construct a
comprehensive DNA
library from all the
microbes in an
ecosystem

A massive study included

50 ocean samples from
different locations
resulted in 6.3billion bp of
sequence and led to
identification of 400 new
bacterial species
 HUMAN GENOME PROJECT:

Supported by NIH and DOE: Though finished, data

analyses will continue for many years
· Project started in 1990 and expected to end in
2005, but ended in 2003
· Sequence genome of E.coli, yeast, Drosophila, &
mouse
• Sequence and analysis of the human genome
working draft was published in February 2001
and April 2003 issues of Nature and Science

http://www.ornl.gov/sci/techresources/Human_Genome/project/journals/journals.html
 HUMAN GENOME PROJECT:

Project Goals

•identify all the 20,000-25,000 genes in human DNA,

•determine the sequences of the 3 billion chemical base pairs
that make up human DNA,
•store this information in databases,
•improve tools for data analysis,
•transfer related technologies to the private sector, and
•address the ethical, legal, and social issues (ELSI) that may
arise from the project.
HUMAN GENOME PROJECT:

· THREE MAPS :

Linkage Map: distance between genetic

markers on chromosome

Whether two genes are linked

(if distance < 0.5 cM)
· THREE MAPS :

Physical Map: No. of nucleotide bases

between genetic markers.

Localize a gene to a particular

region of the chromosome. Go
to freezer and pick up piece of
DNA that contains gene

DNA sequence: ultimate map

sequence of all nucleotides on
all human chromosomes
HUMAN GENOME PROJECT:

· ADVANTAGES:
1. Early detection of diseases (in utero)
2. Replace defective genes for treatment
3. Design drugs to target molecular basis of
disease
· DISADVANTAGE:
1. Understanding the significance of sequence
2. Ethical/ Social implications of early disease
detection/ discrimination in job, marriages,
health insurance etc…3-5% of budget