Recombinant DNA

Technology
Outline
• Genetic material found in bacteria
• Recombinant DNA technology
• DNA cloning
• Gel electrophoresis
• Polymerase chain reaction
• Applications
Definition of terms
• Recombinant DNA
• DNA molecules with sequences from one or more species.

• Recombinant DNA technology

• Combining DNA sequences from 2 or more species.
• Resulting recombinant DNA is introduced into an organism in order to create
new genetic combinations.
• Has applications in basic science, medicine, agriculture and industry.
Genetic material in bacteria
• Bacteria have a nucleoid region
containing their chromosomal DNA
(bacterial chromosome).
• They also have extrachromosomal DNA
known as plasmids.

Source: National Human Genome Research Institute. Available from

https://www.genome.gov/genetics-glossary/Plasmid
Plasmids
• Small, circular, double-stranded DNA molecules separate from a cell’s
chromosomal DNA.
• Contain genes that give bacteria advantages like antibiotic resistance
• When a cell divides, all plasmids get replicated and daughter cells
receive a copy of each plasmid.
• Differ in length depending on species and type of plasmid.
• Have been used by molecular biologists to introduce new genes into
cells.
• These plasmids are introduced into bacterial cells through the process
of transformation.
Plasmids
• Contain an origin of
replication.
• Genes contained depend
on the plasmid type and
the species where it is
found.

Source: Ozyigit. (2012). Crop Production for Agricultural Improvement.

Recombinant DNA technology
• Involves cutting DNA sequences from different organisms and then
pasting them together to get new, novel DNA sequences.
• General steps
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
Recombinant DNA technology
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
Isolation of DNA
• General steps:
• Break open membranes
• Remove cell debris
• Remove proteins
• Remove RNAs
• Precipitate DNA
• Wash DNA
• Remove alcohol used for precipitation and resuspend in buffer
• Examples: Phenol-chloroform extraction, CTAB extraction, magnetic
bead extraction, resin extraction
Isolation of DNA

Source: Phenol Chloroform gDNA extraction. Availble from https://onelab.andrewalliance.com/library/phenol-

chloroform-gdna-extraction-8ZnNJnAb.
Recombinant DNA technology
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
Cutting DNA
• DNA can be fragmented through mechanical shearing.
• gDNA is broken into smaller nucleotide sequences and placed into
libraries.
• Cutting at more specific sites require enzymes known as restriction
endonucleases/enzymes.
Restriction endonucleases
• Enzymes that cut at specific DNA sequences.
• They recognize the sequence and cut the DNA fragment in different
ways
• Sticky end
• Blunt end

Source: Bertero, Brown and Valier. (2017). Methods of Cloning

Restriction endonucleases

A lot of RE
recognition
sites are
palindromic
Recombinant DNA technology
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
Pasting together DNA sequences
• Needs an enzyme called ligase.
• We join a “gene of interest” into a vector
• Gene of interest
• Gene we want to express, gene we want to study, gene we want to clone.
• May be called the insert or the transgene.
• Vector
• Usually a plasmid vector that is used to deliver the gene of interest to the host organism.
• Free nucleic acids are generally degraded in the cell so a vector is needed to protect the
insert.
• The insert is not expressed or replicated without the necessary sequences recognized by
the replication/expression machinery.
Plasmid vectors Multiple Cloning Site

A B

Source: Lodish et al. (2007) Source: Mobi-tech

 Pasting DNA sequences
• Cutting the vector and the gene
of interest with a restriction
endonuclease.
• Sticky-end ligation makes use of
the complementary overhangs
produced by the enzymes.
• Ligase issued to join DNA
fragments together.

Source: DNA Ligation. The University of Queensland. Available https://di.uq.edu.au/community-and-alumni/sparq-

ed/sparq-ed-services/dna-ligation
Pasting DNA sequences
• Blunt-end ligation is more
difficult than sticky-end
ligation.
• DNA ligase attaches the
blunt ends of the vector
and insert.

Source: Integrated DNA Technologies. Available from https://sg.idtdna.com/pages/education/decoded/article/cloning-strategies-

part-3-blunt-end-cloning.
Recombinant DNA technology
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
DNA Cloning
• Clone: cells/organisms which are genetically identical.
• Technique in which specific DNA sequences are inserted into a cloning
vector and is then incorporated into cultured host cells.
• Allows DNA fragments with a particular nucleotide sequence to be
isolated.
• Allows clonal propagation of the recombinant DNA.
Transformation
• The process of taking up DNA which results in the alteration of a cell’s
genetic material
• Makes use of different procedures
• Calcium precipitation
• Heat-shock transformation
• Electroporation
Calcium precipitation
transfection
• Low toxicity
• Simple procedure
• Commonly used for eukaryotic
transfection

Source: Jiang and Chen. (2006). Nature Methods 1:695-700

Heat-shock transformation

1 2 3 4

Source: Thermo Fisher Scientific. Available from https://www.thermofisher.com/ph/en/home/life-

science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-
cloning/transformation/competent-cell-basics.html
Electroporation

1 2 3 4

Source: Thermo Fisher Scientific. Available from https://www.thermofisher.com/ph/en/home/life-

science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-
cloning/transformation/competent-cell-basics.html
Recombinant DNA technology
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
Selection
• How do you know that the cells you plated contain your recombinant
plasmid?
• Selectable markers
• Positive selection markers – presence of the gene is basis for selection
• Antibiotic markers
• Auxotrophy
• Negative selection – loss of a gene product is basis for selection
• Inducible toxins
• Positive and negative selection
• Blas S and Tk gene from HSV
• URA3 selection
Source: Thermo Fischer Scientific. Available from https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-
cloning/cloning/traditional-cloning-basics.html
Recombinant DNA technology
• Isolate DNA
• Cut DNA
• Ligate/paste DNA into vector
• Transformation
• Selection
• Validation
 Validation

Source: Thermo Fischer Scientific. Available from https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-

cloning/cloning/traditional-cloning-basics.html
Plasmid miniprep

Source: Plasmid 101: Restriction cloning. Available from https://blog.addgene.org/plasmids-101-restriction-cloning

Gel electrophoresis
• DNA fragments move through a gel with varying pore size
• DNA is negatively charged so they move down towards the positive
end of the gel
• This technique separates DNA molecules according to size
• Changing the gel concentration changes the pore size and thus you
are able to differentiate even smaller sizes of DNA fragments.
• Agarose is used for routine electrophoresis but polyacrylamide gels
allow for smaller pore sizes.
Agarose gel electrophoresis

Source: DNA gel electrophoresis. Available from http://www.nslc.wustl.edu/courses/bio2960/labs/07DNA/Gel/index.html

 Agarose gel electrophoresis

Source: Agarose Gel Electrophoresis. Available from https://geneticeducation.co.in/agarose-gel-electrophoresis/. Source: Lee et al. (2012). J Vis Exp 20;(62):3923
Restriction enzyme digestion
• You have the vector map and sequence of your plasmid available
• You also know the total size of your insert and your vector so you know the
size of your recombinant (vector + insert)
• Vector only is one size
• Vector + insert is another size
• Vector + random insert is a random size
• Choose a restriction enzyme (you can select more than one) where
you can cut up the plasmid so you have bands of different sizes
• Depending on your enzyme choice, you can predict the band sizes you see in
the gel
 Validation using RE digestion

2700 bp 2700 bp 1500 bp 4200 bp

Source: Access Excellence. Available from http://www.accessexcellence.org/RC/AB/WYW/wkbooks/SFTS/activity6.html?__cf_chl_jschl_tk__=1fbf0af7afc6befa95b98488f13ded0daa405a0f-1604632369-0-

AeABfYAVsyZ0ehOVtc02xSeGmmGjgGA34rtW8CRSoqjLE6j_90rKOELHy39GeCDw6Q9gdGBt0kt2npPkqcbxYm3WAhnejOJQgkgGDf9e4GkLjerykbBdUnQcbjtMMYoh5m32tvFTQg-
HCoKvbNzEo4y_yOyLuQJN1NDZSR0gxaDYbco9X8ZV0ma1_Apm1J_S3yVcmopmLzxW91lJxqY5xedy9Yc5dqWdn3KgqpwGvDjxuyARRkM2MYs1IXQEttW77nsvep6HKMrNOXhlhH_ER3frrb00aTWsNr-77eKUwoIlQCdCKhWCe8mzhiuOdI14TQ
Validation using RE digestion

RE3
RE1
RE1 RE1

RE2
RE4

Expected: 1 band Expected: 2 bands Expected: 3 bands

Validation using RE digestion
RE4
RE1 RE1
RE1

RE2 RE2 RE2

RE3 RE3

Expected: 2 bands Expected: 3 bands Expected: 4 bands

 MW Ladder Vector Vector + insert Vector + other

5000bp
4500bp
4000bp
3500bp

2700 bp 1500 bp 3000bp

2500bp

RE1 2000bp

1500bp
RE2
1000bp

4200 bp 500bp
Polymerase Chain Reaction (PCR)
• Invented by Kary Mullis in 1983
• Allows for exponential amplification of selected DNA fragments
• 3 steps each cycle
• Denaturation
• Annealing
• Elongation/Extension
Denaturation
• Exposing the reaction to 95oC
• Strands melt and separate

Source: Gold Biotechnology. Available from https://www.goldbio.com/goldbios-pcr-overview

Annealing
• Temperature is lowered to 48oC to 70oC.
• Forward and reverse primers anneal to the ssDNA.

Source: Gold Biotechnology. Available from https://www.goldbio.com/goldbios-pcr-overview

Elongation/Extension
• DNA polymerase elongates the
strand from the primers.
• Extension continues at around
72oC.
• 2 copies of one molecule will be
made in each cycle.

Source: Gold Biotechnology. Available from https://www.goldbio.com/goldbios-pcr-overview

Source: Themor Fischer Scientific. Available from https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-
enzymes/pcr-basics.html
 Validation using PCR

Source: Plasmids 101: Colony PCR. Avalable from https://blog.addgene.org/plasmids-101-colony-pcr Source: Ghanbari et al. (2014). Advanced biomedical research 3(7).
 DNA cloning summary

Source: Thermo Fischer Scientific. Available from https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/cloning/traditional-cloning-

basics.html
 Applications of Recombinant DNA technology
• Applications in agriculture

Source: Golden Rice. International rice research institute. Available from https://www.irri.org/golden-rice Source: Cornell Alliance for Science: Available from https://allianceforscience.cornell.edu/blog/2019/07/kenya-field-
trial-shows-bt-cotton-boosts-yields-four-fold/
 Application in medicine

Source: https://www.si.edu/object/nmah_1000970?width=85%25&height=85%25&iframe=true&back_link=1&destination=spotlight/birth-of- Source: Fischer Scientific. Available from https://www.fishersci.fi/shop/products/corning-

biotech/recombinant-drugs basic-fibroblast-growth-factors-bfgf-protein-human-recombinant-3/p-96972
Summary
• Recombinant DNA technology is the process by which different DNA
sequences from different species are joined together in order to
create novel combinations.
• DNA cloning is an essential part of recombinant DNA technology and
it involves clonal propagation of different DNA sequences.
• Involves isolating DNA, cutting DNA and then pasting it onto vectors which
can help facilitate replication in a host cell.
• The results of such manipulations have applications in basic research,
medicine, agriculture and industry.
Recombinant DNA
Technology