Modern cloning techniques

Clones- Organisms that are identical copies, same DNA.

Cutting- Shoots are cut from parent plant, dipped in hormone rooting powder, then in a pot of soil. The HRP stops the
cutting to grow roots resulting in an identical plant. Made possible cause of Stem cells which are able to differentiate
to form different cells and tissues.
Tissue Culture- Cells are taken then placed on nutrient jelly using anti septic technique. Cells will differentiate and
form new plants. (Animals can be cloned with the use of embryo stem cells only.)
Embryo Cloning- Cow is artificially inseminated using bull sperm , embryos grow until an ESC is made. The ESC is
removed from the Uterus and divided and placed into the womb of different cows. Calves will be clones of each
other, not the mother cause bull sperm.
Adult cell cloning - clone an adult animal. Unfertalized egg cell is taken from adult female and nucleus removed. A
nucleus of body cell is taken from another adult and placed into the empty egg cell. Electric shock divides egg cell and
form an embryo. Has same genetic info as adult body cell. Then inerted into a womb of a female surrogate. The clone
is the same as the one who donated the body cell.

BIOTECHNOLOGY Genetic enginnering.

-Utilization of organisms, part of organisms,
biological processes for the benefit of
mankind.
Examples: Lactose Free consumables,
Dough with yeast to make bread, food
items, medicine, hybrid crops, wine
production, golden rice.
Gene technology
- Concerns how genes are expressed, taking advantage of natural
gene variation, modifying genes, and transferring genes to new
hosts
- Genetic engineering such as insulin production and inserting genes
to crops
Vaccines - Prevents diseases; contain inactive particles of viral
coats that has antigen proteins. The immune system produces
antibodies against the antigen proteins, making the body more
prepared.
Edible Vaccines - Inject altered virus into a plant, as it grows, its
cells produce the virus but not infection; have the same effect as
normal vaccines.
Gene Therapy - DNA is introduced into a patient to treat a genetic
disease. (Ex: Mutated gene that causes a genetic disorder such as
cystic fibrosis, a functioning gene is introduced to correct the
disease-causing mutation.) can also block or switch off a gene that
causes cells to malfunction; genes are often introduced through a
viral vector that carries the gene to a patient’s cells directly.
Gene Testing - Done if a genetic disorder is suspected; used to
confirm if you have a specific disorder. (ex. To know a family history
of a genetic condition or belong to group of higher risk; abnormalities
in baby’s genes such as downs and trisomy 18).
- Exists to benefit humans, preventing and curing diseases and food
security.
- The process of using technology to change the genetic makeup of
an organism.
- Manipulation or changing of the DNA of an organism.
- Can be used to make certain microorganisms into factories.
- DNA makes up gene, gene codes different proteins.
- Transferring a donor organism gene to a recipient. the recipient is
transgenic or an GMO to give the transgenic organism have
advantages that the donor naturally has.
**2 MAIN PURPOSES**
- Use of transgenic bacterium to create large volumes of protein such
as Insulin, growth hormone, and vaccines.
- Bt insect toxic gene can be transferred to make toxic corn.
**PRODUCTION OF INSULIN**
- Human insulin gene is cut from the chromosome -> Plasmid is
extracted from bacteria cell -> plasmid is then cut by same restriction
enzymes and leave sticky ends. since same RE were used, the sticky
ends are complementary and allow base pairings. -> Insert the human
insulin gene -> Ligase completed the joining -> plasmid is reinserted
and turns it into a GMO with both human and bacteria DNA. It also
has recombinent DNA. Bacterial DNA is called recombinant DNA as its
recombined with human DNA. -> the bacterial cell is placed in a
fermenter and rapidly reproduces, creating clones with the same
recombinant DNA.
GMO
- GMO’s has had their DNA genetically modified by humans;
changing the genome and characteristics of the organism.
**GM HAPPENS WHEN**
1. Introducing new DNA into a genome using modified
bacteria
2. Introducing new DNA by gene targeting and homologous
recombination
3. Changing regions of a genome using enzymes to cut out
specific parts of the DNA.
-The First and main one relies on bacteria, single celled and
has circular chromosome and plasmids.
-Plasmids are tiny circles of DNA that has one or several
genes; can be passed easily between bacteria.
spider goats - Goats with the spider silk gene, milk will
have spider silk.

DNA Extraction
Clones- Organisms that are identical copies, same DNA.
Cutting- Shoots are cut from parent plant, dipped in
hormone rooting powder, then in a pot of soil. The HRP
stops the cutting to grow roots resulting in an identical
plant. Made possible cause of Stem cells which are able to
differentiate to form different cells and tissues.
Tissue Culture- Cells are taken then placed on nutrient
jelly using anti septic technique. Cells will differentiate and
form new plants. (Animals can be cloned with the use of
embryo stem cells only.)
Embryo Cloning- Cow is artificially inseminated using bull
sperm , embryos grow until an ESC is made. The ESC is
removed from the Uterus and divided and placed into the
womb of different cows. Calves will be clones of each
other, not the mother cause bull sperm.
Adult cell cloning - clone an adult animal. Unfertalized egg
cell is taken from adult female and nucleus removed. A
nucleus of body cell is taken from another adult and placed
into the empty egg cell. Electric shock divides egg cell and
form an embryo. Has same genetic info as adult body cell.
Then inerted into a womb of a female surrogate. The clone
is the same as the one who donated the body cell.
-Glow in the dark UV from jelly fish to see cells in the dark
and has discovered cancer and parkinsons disease also
HIV.
-add nutrition to crops and make them resistant to different
environments and toxins and be toxic to pests.
DNA EXTRACTION
- Cells are lysed using a detergent that disrupts the
plasma membrane.
- Cell contents are treated with protease to destroy
protein and RNAase to destroy RNA.
- Cell debris is pelleted in a centrifuge. The
supernatant liquid containing the DNA is transferred
to a clean tube.
- The DNA is precipitated with ethanol. It forms
viscous strands that can be spooled on a glass rod.
- used for recombinant DNA technology
- Predict virulence of micro organisms
- identification of individuals and paternity
determination.

Polymerase chain reaction RT-PCR in COVID-19 Testing

- Begins with a swab of the nose or throat.
- used to amplify and copy specific segments of
- Saliva, mucus, lung fluid can also be used
DNA.
- The swab of the patient with COVID-19 will contain human cells, virus
- Needs a DNA sample, DNA primers, raw
particles, and other microbes.
nucleotides, and DNA polymerase, goes into a stable
- COVID-19 has RNA as their genetic material; it is surrounded by a
solution and run it through temperature cycles.
nucleocapsid protein within the virus envelope; other proteins are
Step 1. - Denaturation (96C)
embedded with this envelope
-Solution is heated allowing DNA strands to separate
**OBJECTIVE**
Step 2 - Annealing (55C)
- identify part of the viral genome in the patient sample (N gene), which
-Solution is cooled allowing DNA primers to bind
carries directions for making nucleocapsid protein.
Step 3 - Extension (72C)
- There is not enough viral RNA to detect in patient sample so RT-PCR
-DNA polymerase binds and synthesizes new DNA
amplifies many copies of a segment of N gene.
strand.
- Primers(short single-stranded pieces of DNA) recognize unique RNA
(Repeat 30 times over 3 hours to make 1 billion
sequences within the viral genome that bracket the target region of N
copies.
gene.
-Used for DNA mapping, genetic testing, and
- As the primer binds, enzyme reverse transcriptase extends and
ancestry
synthesizes complementary DNA(single stranded copy of viral RNA)
**PCR In forensics**
- As an RNA is removed, 2nd primer binds to the cDNA, then taq
- DNA amolified by PCR and compared to suspects
polymerase extends the 2nd strand to produce double stranded DNA of
- Genetic info put into FBIs DNA database CODIS
target region of viral RNA.
- PCR and CODIS have solved cold cases and
- Essentially same process as PCR
exonerated innocent convicts.
- Each cycle doubles the number of copies of the target region of the
- An important scientific advance.
viral genome.
Gel electrophoresis - virus is detected in 30-45 cycles.
- A fluorescent probe allows real time target DNA detection.
- Technique to separate different DNA fragments based on
their sizes.
- Based on movement of charged molecules when exposed DNA SEQUENCING(SANGER METHOD
to an electrical field. Occurs on agarose gel medium. - Also known as Chain termination sequencing
- Similar to a sieve and works like that. - the DNA must be denatured and converted to single stranded DNA with heat.
- The positive charge moves the fragments. - Separated into template (3-5) strand and complementary strand (5-3).
- Small fragments move ahead farther and faster than - primer is annealed to the template strand
larger fragments. - 4 reaction mixtures as set up with each of them containing a template
- Gel is like a mesh with constant pore size which explains strand with a primer (DNA to be sequenced)
- add DNA polymerase then free nucleotides (dNTP’s) then modified
the separation of the fragments based on size
nucleotides (ddNTP’s)
** REQUIREMENTS **
- DNA is then separated by size through gel electrophoresis
- Casting tray, gel, comb, electrical supply, DNA sample. - all strands with the same sequence cause primer
- Form well like structures to load the DNA mixture on - - each chain depends on ddtp.
- Color the samples to see results with dye.
- treat agarose gel with ethidium bromide that binds with
the dna for us to see the actual DNA
- Can determine the length of the fragments, use a DNA
ladder.
Elution - extraction of DNA fragment from gel, used for
downstream processing.