CHAPTER 18: RECOMBINANT AND BIOTECHNOLOGY

SECTION 18.1

● Recombinant DNA is a DNA molecule that has been in the laboratory using two different sources
of DNA.
● The DNA used for recombination can be from the same organisms (e.g. different chromosomes),
from different members of the same species or from entirely different organisms.
● Restriction enzymes are able to cleave (split) double stranded DNA at particular palindromic
recognition sequences (restriction sites).
● The restriction sites can be either blunt ends or complementary overhangs (short sequences of
single stranded DNA) termed sticky ends. Each restriction enzyme has its own unique sticky end.
● Any two DNA molecules with sticky ends that are complementary can form hydrogen bonds with
one another.
● The sticky ends of the original molecule may bond (anneal) or two different fragments may bond.
● The fragments are initially held together by only a few weak hydrogen bonds, since there is a
missing phosphodiester bond ( a “nick”) in each strand of the double helix.
● The nicks can be sealed by their DNA ligase to catalyze the formation of the strong , covalent,
phosphodiester bonds between adjacent nucleotide in each of the strand, joining them to form a
single, larger molecule
 ● Restriction enzymes and DNA ligase are able to create recombinant DNA.
● Herbert Boyer and their colleagues created recombinant bacterial plasmids.
● They isolated two different plasmids.Each of the two plasmids contained a different
antibiotic resistance gene. The scientist cut the two plasmids with restriction enzymes,
mixed the fragment together , and then used DNA ligase to rejoin the DNA molecule,
creating recombinant plasmids.
● The recombinant plasmids of this ligation reaction were then inserted into new E. coli
cells ,and the cells were grown in the presence of both antibiotics.
● The transformed bacteria could grow on the medium, and the research were able to show
that a single recombinant plasmid containing the genes for resistance to both antibiotics

EXPERIMENT

● With discovery of restriction enzymes and DNA ligase it becomes possible to combine
DNA fragments from different sources in the laboratory.
● The experiment by stanley cohen and herbert boyer completely changed the scope of
genetic research, increasing our knowledge of gene structure and function and ushering in
the field of biotechnology.

●
● Methods based on the polymerase chain reaction (PCR) allow the joining of any two
DNA molecules.
SECTION 18.2

● One of reason for making recombinant DNA is to clone to produce many identical copies
of a particular gene or other DNA sequence
● Recombinant DNA is cloned by inserting it into host cell in a process known as
Transformation or transfection
● A host cell or organism that contains recombinant DNA is referred to as a transgenic cell
or organism , and the non- native DNA is called a transgene.

SELECTIVE GENETIC MARKERS ARE USED TO IDENTIFY HOST CELLS

CONTAINING RECOMBINANT DNA

● In order to isolate and reproduce only transgenic cells - selectable marker genes- such as
genes that confer resistance to antibodies, are often included as part of the recombinant
DNA molecule.
● When an antibiotic resistance gene is used as the selectable marker, the cells from the
transformation experiment are grown in the presence of the antibiotic; the antibiotic
kills all non transgenic cells, leaving only the transgenic cells. Antibiotic resistance
genes were the selectable markers used in Cohen and Boyer’s experiment.

GENES CAN BE INSERTED INTO PROKARYOTIC OR EUKARYOTIC CELLS

● Any cells or organisms can host the introduction of recombinant DNA.

- Bacteria are easily manipulated in the laboratory. Bacteria contain plasmids,
which are easily manipulated to carry recombinant DNA into the cell.
- Yeasts are used as eukaryotic hosts for recombinant DNA studies. The advantage
of using yeasts include their rapid cell division, ease of growth in the laboratory,
and relatively small genome size.

● For multicellular organisms, it is possible to produce transgenic organisms by

transforming a single cell. Multicellular sexual organism pass thorugh a single celled
zygote stage that develops into an adult organism containing many thousand to many
trillions of cells.
● One method for creating transgenic individuals involves creating transgenic zygotes by
injecting fertilized eggs with a transgene.The transgenic zygotes then develop into
transgenic organisms with every cell transgenic.
 ● An alternative method for obtaining transgenic multicellular organisms is to use cell
culture. The cultured cells must be totipotent stem cells - cells that have the ability to
develop into any other cell type.
● Transformation is performed in culture, and transgenic cells are selected and allowed to
produce large numbers of daughter cells.
● This result of injecting transgenic cells is an embryo that is a mixture of original cells and
transgenic cells.
● If transgenic cells give rise to gamete in the adult animal, then the resulting zygotes will
have the transgene and will develop into offspring that have the transgene in all of their
cells.

INSERTION DNA IS USUALLY INTEGRATED INTO THE HOST CHROMOSOMES

● Some prokaryotic cells can take up intact DNA molecules from the environment which makes
transformation quite easy.
● Most cells cannot be used in transformation and so various laboratory methods have been devised
to get DNA into cells.
● Cells can be chemically treated to make their outer membrane more permeable,and then mixed
with the DNA so that it can diffuse into the cells.
● Another approach is called - electroporation- a short electric shock is used to create temporary
holes in the membrane through which the DNA can diffuse into the cell.
 ● If the new DNA is to be replicated, it must become part of a segment of DNA that contains an
origin of replication. Such a DNA molecule is called a replicon or replication unit.
● There are two general ways in which the newly introduced DNA can become part of a replicon
within the host cell
- It can be spliced into a host chromosome directly.
- It can enter the host cell as part of a carrier DNA sequence, called a vector, and can then
either integrate into a host chromosome or be replicated from its own origin of
replication.

PLASMIDS AS VECTORS

Several types are used to get DNA into cells.

● Plasmids are generally small,circular DNA molecules that replicate autonomously in many
prokaryotic cells.
● Several characteristics make plasmids useful as transformation vectors:
- Plasmids are relatively small and are therefore easy to manipulate in the laboratory.
- Plasmids often have one or more restriction enzyme recognition sequences that each occur
only once in the plasmid sequence.This makes it easy to insert new DNA into the prokaryotic
plasmid before it is used to transform host cells.
- Many plasmids contain genes that confer resistance to antibiotics, which can serve as
selectable markers.
- Plasmids have a bacterial origin of replication (ori ) and can replicate independently if the host
chromosomes.it is not common for a bacterial cell to contain hundreds of copies of a
recombinant plasmid. For this reason, the power of bacterial transformation to amplify a gene
is extraordinary.

● A plasmid used as a vector in the laboratory has been extensively altered to include convenient
features.

- Multiple unique restriction sites for cloning purposes

- An origin of replication that will function in a particular type of host cell.
- One or more selectable markers

PLASMIDS AS VECTOR FOR PLANTS

● An important vector for carrying new DNA into many types of plant is a plasmid found
in the bacterium Agrobacterium tumefaciens.
 ● When the bacterium infects a plant cells, a region of the Ti plasmid called the T DNA is
inserted into the host plant cell, where it become incorporated into one of the plant’s
Chromosomes.
● The Ti plasmid carries the genes needed for this transfer and incorporation of the T
DNA.
● The T DNA carries genes that are then expressed by the host cell, causing the growth of
tumor and the production of specific sugar that the bacterium uses as a source of energy.
● The recombinant Ti plasmids are first used to transform agrobacterium cells from which
the original Ti plasmids have been removed. Then Agrobacterium cells are used to infect
plant cells and transfer the T DNA, now containing genes placed there by the research to
the host cell chromosomes.

VIRUSES AS VECTORS

● Both prokaryotic and eukaryotic viruses are used as vectors for eukaryotic DNA.
All of which are injected into a host cell during infection.
Base pairs can be deleted and replaced with DNA from another organisms which then gets
replicated along with virus DNA.

REPORTER GENES HELP SELECT OR IDENTIFY HOST CELLS CONTAINING

RECOMBINANT DNA

● One of the ways to use selectable markers. Selective markers are one type of reporter
gene, which is any gene whose expression is easily observed. Here are several types of
reporter genes :

- An antibiotic gene in a plasmids or other vector allows the detection of

transformed host cells grown in the presence of the selected antibiotic.
- If the host cells are normally sensitive to the antibiotic, they will grow on
medium containing the antibiotic only if they have been transformed by the
vector. This approach is used in the selection of transgenic prokaryotic and
eukaryotic cells, including those of plants and animals.
- In some cases a plasmid contains two antibiotic resistance genes and one of
them is used for cloning. For example, suppose the plasmid in was used to
clone a piece of foreign DNA at the BamHI site. Transformation could be
selected for using ampicillin. The resulting bacteria could then be tested
(screened) for resistance to tetracycline. Bacteria that were resistant to
ampicillin and sensitive to tetracycline would have the plasmid containing a
piece of foreign DNA.
 - The lacZ gene can be used to screen transformed colonies to see if their
plasmid contains an inserted fragment of foreign DNA.
- GFP has also been modified to emit other colors when exposed to ultraviolet
light, allowing multiple reporters to be used at the same time.
- Both lacZ and GFP are widely used as reporter genes in other situations
besides cloning. The protein-coding sequence of one of these reporter genes
can be attached to promoters or other regulatory sequences from other genes to
study how these sequences regulate gene expression under different conditions
or in different tissues of a transgenic multicellular organism.
- Overall : Reporter genes such as lacZ and GFP can be used for an insert in a
cloning vector, measure expression, or determine protein location.

How are numerous transgenic individuals produced from one ?

Producing numerous clones of multicellular,sexual organism involves

producing inbred, genetically uniform individual that are homozygous for the
transgene.

The cloning site is used for inserting foreign DNA. the origin of replication allows the vector to
make copies of itself. The selectable marker for selection of the vector in the host.

SECTION 18.3 DNA FOR STUDY IS OBTAINED IN A VARIETY

OF WAYS.

DNA for study can come from a library

● A Genomic library is a collection of DNA fragments that make up the entire genome of an organism.
● Restriction enzyme digestion or other means can be used to break chromosomes into small pieces.
● Each fragment is then cloned by inserting it into vector which is then taken up by a host cell.
● Proliferation of a single such transformed cell produces a colony of cell, each of which many copies of
the same fragment of DNA.
● Many clones are required to make a genomic library for an entire genome.

cDNA is made from mRNA transcripts

● The one that includes only the genes transcribed in a particular tissue at a particular point in time can
be made from complementary DNA or cDNA.
● Making cDNA involves isolating mRNA from cells then making cDNA copies of that mRNA by
complementary base pairing in a process called reverse transcription.
● Reverse transcription process is catalyzed by the reverse transcriptase enzyme.
 ● The collection of cDNA from a particular time in the life cycle of an organism is called a
cDNA library because mRNA don’t last long in the cytoplasm.

DNA can be amplified using PCR or can be synthesized artificially.

● PCR : Using PCR it is possible to generate thousands to millions of copies of a particular section
of DNA from a very small amount of DNA. PCR is a common tool used in medical and biological
research labs.
● In this case, mRNA is isolated from an organism or tissue, and reverse transcriptase is used to
make cDNA from the mRNA. Then PCR is used to amplify a specific sequence directly from the
sample of cDNA. This procedure, called RT-PCR.
● Artificial DNA can be synthesized using organic chemistry to link nucleotides together in a
specified sequence.
● The synthesis of artificial DNA does not require a template, so DNA with any sequence can be
made.
● Plasmids containing synthetic transposon DNA have been used to transfect host cells to inactivate
genes one by one and give insight into the minimal genome.
● DNA for study can be obtained from genomic libraries, cDNA libraries, and PCR, or it can be
artificially synthesized.

SECTION 18.4 : SEVERAL TOOLS ARE USED TO STUDY THE

FUNCTION OF DNA.

GENES OF INTEREST CAN BE IDENTIFIED BY GENETIC MAPPING OR BY STUDYING

GENE EXPRESSION

● To identify distinct patterns of gene expression, scientists could isolate mRNA from cells and
measure the amount of each gene’s mRNA one gene at a time by hybridization or RT-PCR.
● But that would be expensive and time-consuming. It is far simpler to do these hybridizations
for every gene in a single step, and that is made possible with DNA microarray technology.
● use of DNA microarrays for single nucleotide polymorphism (SNP) genotyping . For RNA
expression, each microscopic spot contains thousands of copies of a particular
oligonucleotide of 20 or more bases that is the sequence of an exon from a protein-coding
gene in the genome. Each oligonucleotide can hybridize with only one RNA sequence and
thus is a unique identifier of expression of that gene. If a gene is highly expressed, there will
 be many RNA molecules in the sample to hybridize to the corresponding spot on the
microarray.

The function of a gene can be determined by modifying its expression

post-transcriptionally

● Gene expression is sometimes regulated by the production of double-stranded RNA molecules

that are processed to produce short, single-stranded microRNAs (miRNAs) and small interfering
RNAs (siRNAs). These miRNAs and siRNAs are complementary to specific mRNA sequences
and combine with proteins to form an RNA-induced silencing complex (RISC). When miRNAs
and siRNAs are hybridized (bound) to their target mRNAs, the RISC inhibits translation
(miRNAs) or causes degradation (siRNAs) of the mRNA.
● A related method uses synthetic single-stranded RNA sequences complementary to target
mRNAs that prevent translation by hybridizing to the mRNA. The synthetic RNA is called
antisense RNA because it binds by base pairing to the “sense” bases on the mRNA
 Genes can be activated and altered using CRISPR technology

Each spacer is a fragment of DNA from a virus that previously infected the
cell but did not end up killing its host. The spacer sequences thus provide
“genetic mug shots” that inform the cell carrying them what viruses to look
out for. When a virus carrying a sequence similar to the spacer DNA injects
its DNA into the cell, the unit—CRISPR and spacer—is transcribed into
RNA, to which a second short RNA attaches. This RNA complex then
performs the following two functions:

■ Because the spacer sequence is complementary to part of the viral genome, it attaches
to it by base pairing.
■ The complex binds a protein called CAS9, which is a nuclease that creates a
double-strand break of the invading virus DNA at the recognized sequence,
destroying its ability to produce new viruses and thereby protecting the host cell.
■ CRISPR can also be used to knock down expression of a gene. This approach uses a
CAS9 that has been modified so that it can no longer cut DNA. The inactivated CAS9
binds both the guide RNA and DNA and can thus be directed to a gene. Once bound,
the inactivated CAS9 physically prevents transcription of the gene, even though it
does not cut the DNA.

■ CRISPR allows a gene to be knocked out, knocked down, or altered.