This document discusses gene synthesis techniques and blotting methods. It provides details on: 1) The first chemical synthesis of genes in the 1970s, including a gene for yeast tRNA and bacterial tRNA. 2) Methods for artificially synthesizing genes using oligonucleotides and ligating DNA fragments. 3) Techniques for analyzing DNA, RNA, and proteins - Southern blotting detects DNA, Northern blotting detects RNA, and Western blotting detects proteins.

1. Organochemical gene
synthesis, Blotting
techniques- Southern,
Northern and Western
Blotting Promila Sheoran
Ph.D. Biotechnology
GJU S&T Hisar

2. •Artificial gene synthesis, sometimes known as DNA printing is a method
in synthetic biology that is used to create artificial genes in the laboratory.
• Currently based on solid-phase DNA synthesis, it differs from molecular
cloning and polymerase chain reaction (PCR) in that the user does not have to begin
with preexisting DNA sequences.
• Therefore, it is possible to make a completely synthetic double-stranded DNA
molecule with no apparent limits on either nucleotide sequence or size.
• The method has been used to generate functional bacterial or yeast chromosomes
containing approximately one million base pairs.

3. •For the first time in 1955, Michelson chemically synthesized a dinucleotide in
laboratory.
•Later on in 1970, Har Govind Khorana and K.L. Agarwal for the first time chemically
synthesized gene coding for tyrosine tRNA of yeast.
•For the synthesis of tRNA and rRNA there are specific genes. However, genes of tRNA
are the smallest genes containing about 80 nucleotides.
• In 1965, Robert W. Holley and coworkers worked out first the molecular structure of
yeast alanine tRNA.
• This structure lent support to Khorana in deduction of structure of the gene. A gene
is responsible for encoding mRNA, and mRNA for polypeptide chain. If the structure of
a polypeptide chain is known, the structure of mRNA from genetic code dictionary and
in turn the structure of gene can easily be worked out.
•There are two approaches for artificial synthesis of the gene, by using chemicals and
through mRNAs.

4. Synthesis of a Gene for Yeast alanine tRNA
•As mentioned earlier that the molecular structure of yeast alanine tRNA was
worked out by R.W. Holley and coworkers in 1965 which helped Khorana to deduce
the structure of alanine tRNA.
•They found out that yeast alanine tRNA contains 77 base pairs.
•It was very difficult to assemble 77 base pairs of nucleotides in ordered form.
Therefore, they synthesized chemically the short deoxynucleotide sequences which
was joined by hydrogen bonding to form a long complementary strand.
•By using polynucleotide ligase the double stranded pieces were produced. The
complete procedure of synthesizing gene for yeast alanine tRNA is discussed in the
following steps.

5. (i) Synthesis of Oligonucleotides.
• In the first approach, fifteen oligonucleotides ranging from
pentanucleotide (i.e.oligodeoxynucleotide of five bases) to an
icosanucleotide (i.e.oligodeoxynucleotide of twenty bases) were synthesized.
•The chemical synthesis was brought about through condensation between the - OH
group at 3' position of one deoxynucleotide and the - PO4 group at 5' position of the
second deoxynucleotide.
•All other functional groups of deoxyribonucleotides not taking part in condensation
processes were protected so that the condensation could be brought about.

6. The group in base or sugar to
be protected
Protected by Protecting group removed
by
A. -NH2 group (base)
(i) deoxyadenosine Benzyl group Ammonia
(ii) deoxycytidine Anisoyl group Ammonia
(iii) deoxyguanosine Isobutyl group Ammonia
B. -OH group (sugar)
(i) -OH group at
5' position of first nucleotide
Monomethoxy trityl
group
Acid
(ii) -OH group at 5' position of
growing chain
Cyanoethyl Alkali
(iii) -OH group at 3' position Acetyl group Alkali
Table 2.5. Different protective groups of nucleotides and their removal

7. •Finally, condensation between the groups of two, three or four nucleotides was
brought about.
•The receiving segment had a free 3'-OH group and a protected 5'-OH group,
whereas the incoming segment had a free 5'-OH group and a protected 3'-OH group.
• After each addition, the protective group at the 3' end had removed so that free 3'-
OH group could receive another segment.
(ii) Synthesis of three duplex fragments of a gene.
• By using 15 single stranded oligonucleotides, three large double stranded DNA
fragments were synthesized.

8. These three fragments contained
(i) segment of A having the first 20 nucleotides with the nucleotides 17-20 as the single
stranded,
(ii) segment B consisting of nucleotides 17-50 with the nucleotides 17-20 and 46-50 as
the single stranded, and
(iii) segment C containing the nucleotides 46-77 with the single stranded region 46-50.

9. (iii) Synthesis of a gene from three duplex fragments of DNA
•The three segments (A,B,C) synthesized is above were joined by using the enzyme
polynucleotide ligase to produce the complete gene for alanine tRNA.
The joining of the three fragments was done by any of two following methods:
(a) In one approach, fragment A was joined to B by taking advantage of overlapping in
nucleotide residues 17-20. Then, the fragment C was added with the overlap in
nucleotides 46- 50. Thus, a complete double stranded DNA with 77 base pairs was
prepared.
(b) In the second approach, the fragment B was joined to C. At the end the fragment A
was added to nucleotide residues 17-20 to obtain the complete gene for alanine tRNA.
•Khorana et. al. (1970) prepared this gene in vitro which was used for future work. They
found that alanine tRNA gene replicated and transcribed into tRNA just like the natural
gene

10. Artificial Synthesis of a Gene for Bacterial tyrosine tRNA
•In 1975, Khorana and co-workers completed the synthesis of a gene for E. coli tyrosine
tRNA precursor.
•E. coli tRNA precursors are formed from the larger precursors. The tyrosine tRNA
precursor has 126 nucleotides. They synthesized the complete sequence of DNA duplex
coding for tyrosine tRNA precursor of E. coli, and promoters are terminator genes.
• Though these segments are not the proper structural gene yet are the regions
involved in its regulation. Twenty six small oligonucleotide DNA segments giving rise to
tRNA precursor were synthesized which were arranged into six double stranded
fragments each containing single stranded ends.
• These six fragments were joined to give rise complete gene of 126 base pairs for
tyrosine tRNA precursor of E. coli.

11. •Khorana (1979) completely synthesized a biologically functional tyrosine tRNA
suppressor gene of E. coli which was 207 base pairs long and contained
(i) a 51 base pairs long DNA corresponding to promoter region,
(ii) a 126 base pair long DNA corresponding to precursor region of tRNA,
(iii) a 25 base pair long DNA including 16 base pairs contained restriction site
forEcoRI.
•This complete synthetic gene was joined in phage lambda vector which in turn was
allowed to transfect E. coli cells.
•After transfection phage containing synthetic gene was successfully multiplied in E.
coli.

12. •Khorana (1979) made the phosphodiester approach for synthesizing the
oligonucleotides of the biologically active tRNA.
•The demerits of this approach are:
(i) the completion of reaction in long time,
(ii) rapidly decrease in yield with the increase in chain length, and
(iii) time taking procedure of purification.

13. Artificial Synthesis of a Human Leukocyte Interferon Gene
•Interferons are proteinaceous in nature produced in human to inhibit viral infection.
These are of three types secreted by three genes i.e.
(i) leukocyte interferon gene (IFN-oc gene),
(ii) fibroblast interferon gene (IFN-p gene), and
(iii) immune interferon gene (IFN-γ gene).
•In 1980, Weismann and co-workers published the nucleotide sequence of IFN-a gene.
Taking advantage of this information Edge et al. (1981) successfully synthesized the total
human interferon gene of 514 base pairs long.
•Edge et al. (1981) made the phosphotriester approach in artificially synthesizing 67
oligonucleotides of 10-20 nucleotide residue long segment.
•The phosphotriester approach overcomes some of the demerits of phosphodiester
approach by blocking the function of each internucleotide phosphodiester during the
process of synthesis.
•A completely protected mononucleotide containing a fully masked 3' phosphate
triester group is used in this method.

14. •Coupling of initial nucleotide onto a polyacrylamide resin was done to which
further nucleotides in pairs were added.
• In this way 66 oligonucleotides of 14-21 nucleotide residues were first
synthesized.
•These were arranged in predetermined ways and joined chemically. The 514
base" pairs long IFN-a gene contained the initiation and termination signals.
•Edge et. al. (1981) incorporated the artificially synthesized gene into a plasmid
through biotechnological technique.
•The recombinant plasmid was transferred into E.coli cells which expressed oc-
interferon. This technique now-a-days is being adopted to produce interferon
commercially.

15. Blotting Techniques—Southern, Northern, Western Blotting
•These are techniques for analyzing cellular macromolecules: DNA, RNA, and
protein.
•Southern Blotting technique was developed by E.M. Southern in 1975.
•Northern blotting technique was developed by Alwine and colleagues in 1979.
•Western blotting technique was developed by Towbin in 1979.

16. Complementarity and Hybridization
•Molecular searches use one of several forms of complementarity to identify the
macromolecules of interest among a large number of other molecules.
•Complementarity is the sequence-specific or shape-specific molecular recognition
that occurs when 2 molecules bind together. For example: the 2 strands of a DNA
double-helix bind because they have complementary sequences; also, an antibody
binds to a region of a protein molecule because they have complementary shapes.
•Complementarity between a probe molecule and a target molecule can result in
the formation of a probe-target complex. This complex can then be located if the
probe molecules are tagged with radioactivity or an enzyme. The location of this
complex can then be used to get information about the target molecule.

17. •In solution, hybrid molecular complexes hybrids of the following types can exist:
1. DNA-DNA. A single-stranded DNA (ssDNA) probe molecule can form a double-
stranded, base-paired hybrid with a sDNA target if the probe sequence is the
reverse complement of the target sequence.
2. DNA-RNA. A single-stranded DNA (ssDNA) probe molecule can form a double-
stranded, base-paired hybrid with an RNA target if the probe sequence is the
reverse complement of the target sequence.
3.Protein-Protein. An antibody probe molecule can form a complex with a target
protein molecule if the antibody’s antigen binding site can bind to an epitope
(small antigenic region) on the target protein. In this case, the hybrid is called an
“antigen-antibody complex” or “complex” for short.

18. Blots are named for the target molecule.
•Southern blot. DNA cut with restriction enzymes-probed with radioactive DNA.
•Northern blot. RNA-probed with radioactive DNA or RNA.
•Western blot. Protein-probed with radioactive or enzymatically tagged antibodies.

19. In general, the process has the following steps:
1. Gel electrophoresis
2. Transfer to solid support
3. Blocking
4. Preparing the probe
5. Hybridization
6. Washing
7. Detection of probe-target hybrids

20. Gel Electrophoresis
•This is a technique that separates molecules on the basis of their size. The gel is cast
soaked with buffer.
•The gel is then set up for electrophoresis in a tank holding buffer, with electrodes to
apply an electric field.
•The pH and other buffer conditions are arranged so that the molecules being
separated carry a net (–) charge so that they will be moved by the electric field from
left to right.
•As they move through the gel, the larger molecules will be held up as they try to
pass through the pores of the gel, while the smaller molecules will be impeded less
and move faster.
• This results in a separation by size, with the larger molecules nearer the well and
the smaller molecules farther away.

22. Preparing DNA for Southern Blots
DNA is first cut with restriction enzymes and the resulting double-stranded
DNA fragments have an extended rod conformation without pretreatment.
Preparing RNA for Northern Blots
Although RNA is single-stranded, RNA molecules often have small regions that can
form base-paired secondary structures. To prevent this, the RNA is pretreated with
formaldehyde.
Preparing Proteins for Western Blots
Proteins have extensive 2' and 3' structures and are not always negatively charged.
Proteins are treated with the detergent SDS (sodium dodecyl sulfate), which removes
2' and 3' structure and coats the protein with negative charges. If these conditions
are satisfied, the molecules will be separated by molecular weight, with the high-
molecular-weight molecules near the wells and the lowmolecular- weight molecules
far from the wells.

23. Different stains and staining procedures are used for different classes of
macromolecules:
Staining DNA. DNA is stained with ethidium bromide (EtBr), which binds to nucleic
acids. The DNA-EtBr complex fluoresces under UV light.
Staining RNA. RNA is stained with ethidium bromide (EtBr), which binds to nucleic
acids. The RNA-EtBr complex fluoresces under UV light.
Staining Protein. Protein is stained with Coomassie Blue (CB). The protein- CB complex
is deep blue and can be seen with visible light.

24. Transfer to Solid Support
•After the DNA, RNA, or protein has been separated by molecular weight, it must be
transferred to a solid support before hybridization. (Hybridization does not work well
in a gel.)
•This transfer process is called blotting and is why these hybridization techniques are
called blots.
•Usually, the solid support is a sheet of nitrocellulose paper (sometimes called a filter
because the sheets of nitrocellulose were originally used as filter paper), although
other materials are sometimes used.
•DNA, RNA, and protein stick well to nitrocellulose in a sequence-independent
manner.

25. The DNA, RNA, or protein can be transferred to nitrocellulose by Capillary blotting,
where the molecules are transferred in a flow of buffer from wet filter paper to dry
filter paper:

26. Blocking
•At this point, the surface of the filter has the separated molecules on it, as well as
many spaces between the lanes, etc., here no molecules have yet bound.
• If we added the probe directly to the filter now, the probe would stick to these
blank parts of the filter, like the molecules transferred from the gel did.
• This would result in a filter completely covered with probe, which would make it
impossible to locate the probe-target hybrids.
•For this reason, the filters are soaked in a blocking solution, high contains a high
concentration of DNA, RNA, or protein. This coats the filter and prevents the probe
from sticking to the filter itself.

27. Hybridization
•In all 3 blots, the labeled probe is added to the blocked filter in buffer and incubated
for several hours to allow the probe molecules to find their targets.
Washing
•After hybrids have formed between the probe and target, it is necessary to remove
any probe that is on the filter that is not stuck to the target molecules.
•Because the nitrocellulose is absorbent, some of the probe soaks into the filter and
must be removed. If it is not removed, the whole filter will be radioactive and the
specific hybrids will be undetectable.To do this, the filter is rinsed repeatedly in
several changes of buffer to wash off any unhybridized probe.
Note
In Southerns and Northerns, hybrids can form between molecules with similar but
not necessarily identical sequences (For example, the same gene from 2 different
species). This property can be used to study genes from different organisms or genes
that are mutated.

28. Detecting the Probe-Target Hybrids
At this point, you have a sheet of nitrocellulose with spots of probe bound wherever
the probe molecules could form hybrids with their targets. The filter now looks like a
blank sheet of paper—you must now detect where the probe has bound.
Autoradiography
If the probe is radioactive, the radioactive particles that it emits can expose x-ray film.
If you press the filter up against x-ray film and leave it in the dark for a few minutes to
a few weeks, the film will be exposed wherever the probe bound to the filter. After
development, there will be dark spots on the film wherever the probe bound.
Enzymatic Development
If an antibody-enzyme conjugate was used as a probe, this can be detected by
soaking the filter in a solution of a substrate for the enzyme. Usually, the substrate
produces an insoluble colored product (a chromogenic substrate) when acted upon
by the enzyme. This produces a deposit of colored product wherever the probe
bound.

30. Applications of Southern blotting technique
•To identify DNA in a specific DNA sample.
•To isolate desired DNA for construction of Rdna
•To identify deletions, mutations and gene arrangements.
•Diagnosis of infectious diseases.
•In DNA fingerprinting
Paternity and Maternity testing
Criminal identification and forensics
Personal identification

31. Applications of Northern blotting technique
•Detecting a specific mRNA in a sample.
•In gene expression studies.
•Used in the screening of recombinants by detecting the mRNA produced by the
transgene.
•In disease diagnosis.

32. Applications of Western blotting technique
•Highly sensitive method to detect a specific protein in a very low quantity.
•Used in clinical diagonsis.
•Quantifying a gene product.

33. Thank You