Recombinant DNA technology involves combining DNA molecules from different sources that are not normally found together. It begins with restriction enzymes that cut DNA at specific sequences and DNA cloning vectors. DNA fragments are joined to the vector which is then placed into a host cell to replicate many copies. The copies are clones that can be studied. Recombinant DNA is used to isolate, replicate, and analyze genes. Key tools are restriction enzymes, plasmids, and genomic and cDNA libraries which allow screening for genes of interest.

1. Genetic engineering and
recombinant DNA technology

2. Cont----
• Recombinant DNA refers to the joining of DNA
molecules, usually from different biological
sources, that are not found together in nature
• Recombinant DNA Technology Began with Two
Key Tools: Restriction Enzymes and DNA
Cloning Vectors

3. Cont----
• The basic procedure for producing
recombinant DNA involves
– generating specific DNA fragments using
restriction enzymes
– joining these fragments with a vector
– transferring the recombinant DNA molecule to a
host cell to produce many copies that can be
recovered from the host cell

4. Cont----
• The recovered copies of a recombinant DNA
molecule are referred to as clones and can be
used to study the structure and orientation of
the DNA
• Recombinant DNA technology is used to
isolate, replicate, and analyze genes

5. Restriction endonucleases
• Restriction Enzymes Primarily found in
bacteria (they use these for defense)
• 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

6. Cont----
• 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

7. Cont----
• 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

8. Cont----
• Enzymes that produce sticky ends are
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

13. Vectors
• Vectors are carrier DNA molecules that can
replicate cloned DNA fragments in a host cell
• Vectors must be able to replicate
independently and should have several
restriction enzyme sites to allow insertion of a
DNA fragment
• Vectors should carry a selectable gene marker
to distinguish host cells that have taken them
up from those that have not

14. Plasmid
• 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
• Can replicate independently of chromosome
• Can be used as vectors – pieces of DNA that
can accept, carry, and replicate other pieces of
DNA

16. How to Isolate the Gene of Interest
• Use Reverse Transcriptase to make the gene
of Interest
1. Isolate mRNA for the gene product of
interest (e.g. Insulin mRNA)
2. Use Reverse Transcriptase to produce cDNA
(complementary DNA)
3. Use PCR to clone the cDNA
3. Separate the synthetic gene of interest by
electrophoresis

18. Cont----
Method #2
1. Determine the primary structure (i.e. the amino
acid sequence) of the protein of interest (e.g.
insulin) with an automated protein sequencer
2. Use table of codons to determine the mRNA
sequence
3. Synthesize the mRNA in the lab
4. Use Reverse Transcriptase to produce cDNA and
PCR to clone the cDNA (as before)
5. Separate the synthetic gene of interest by
electrophoresis

19. How to Create Recombinant DNA
1. Digest a plasmid vector with a restriction
enzyme (e.g. EcoRI) at a single site to
produce two sticky ends.
2. Digest human DNA with EcoRI to produce
pieces with the same sticky ends
– Use Human DNA or cDNA copied from
mRNA using reverse transcriptase from
retroviruses.

20. Cont----
1. Mix the two samples and allow to hybridize.
• Some plasmids will hybridize with pieces
of human DNA at the EcoRI site.
2. DNA ligase is used to covalently link the
fragments.

22. Insertion of Recombinant Plasmids
into Prokaryotic cell

24. Identification of cells containing
plasmids
• Cells containing plasmids contain the
ampicillin resistance gene
• Grow cells on medium containing ampicillin
• If the cells contain plasmid they can grow and
form colonies on the media if they do not, no
colony will be formed on the media.

25. Cont----

27. 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

28. Cont----
–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!

29. cDNA Libraries
– mRNA from tissue of interest is isolated
– enzyme reverse transcriptase catalyzes
synthesis of complementary single stranded
DNA from mRNA
– It is Called complementary DNA (cDNA)
because it is an exact copy of the mRNA
– The mRNA is degraded either with an
enzyme or alklaline solution

30. Cont----
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

31. Cont----
–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

32. Cont----
–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

35. Cont----