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Recombinant DNA Technology Lecture

Recombinant DNA technology involves combining DNA sequences from different organisms. Key steps include isolating DNA, cutting DNA with restriction enzymes, pasting the DNA fragments into a vector, transforming host cells, selecting for recombinant cells using markers, and validating the recombinant DNA. Validation techniques include restriction enzyme digestion followed by agarose gel electrophoresis to check for DNA fragments of the expected sizes. This confirms that the intended DNA sequence was correctly inserted into the vector.
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0% found this document useful (0 votes)
167 views50 pages

Recombinant DNA Technology Lecture

Recombinant DNA technology involves combining DNA sequences from different organisms. Key steps include isolating DNA, cutting DNA with restriction enzymes, pasting the DNA fragments into a vector, transforming host cells, selecting for recombinant cells using markers, and validating the recombinant DNA. Validation techniques include restriction enzyme digestion followed by agarose gel electrophoresis to check for DNA fragments of the expected sizes. This confirms that the intended DNA sequence was correctly inserted into the vector.
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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

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