UNIVERSITY OF CEBU-BANILAD

COLLEGE OF MEDICAL TECHNOLOGY

(Medical Laboratory Science)

COURSE CODE: MLS 101

DESCRIPTIVE TITLES: PRINCIPLES OF MEDICAL LABORATORY PRACTICE 1
( Intro to Medical Laboratory Science, Laboratory Safety and Waste Management )

F. GENETIC ENGINEERING
Genetic engineering involves methods, techniques, and procedures used in gene manipulation. The
oldest of these is the recombinant DNA (rDNA) technique or gene splicing where DNA of the desired
gene is inserted into the DNA of another organism to produce the desired gene. The first rDNA was
created by molecular biologist Paul Berg in 1972 from the cancer-causing monkey virus SV40 and the
virus lambda. This is marked the birth of the field of genetic engineering.

Other procedures in genetic engineering:

1. Microinjection - a gene from another organism is injected into the recipient cell where it
automatically enters into the nucleus and incorporates itself into the host cell’s genetic materials and
replicates.

2. Bioballistics - involves the insertion of a small silver particles coated with genetic material into
recipient cells.

3. Electroporation and chemical poration - pores are created using weak electric currents or
chemicals, respectively, in the cell surface to transfer genes.

History of genetic engineering:

1972 - gene therapy was first conceptualized.

1974 - Mouse is commonly used in research as an animal model. The first genetically modified
mouse was created by Rudolf Jaenisch. Transgenic mice contain articially- introduced genetic
material in each of their cells. They are used to determine overexpression or misexpression of a
particular protein and to elucidate the DNA sequences that regulate the expression of a genes.

1979 - the method of producing insulin using genetic engineering was discovered. This the first
transgenic drug in biopharming.

1990 - the Human Genome Project began, which discovered an estimated 20,000 to 25,000 human
genes and determined the complete sequence of the 3 billion DNA subunits.

1991 - first trial on humans was conducted.

1997- first cloned animals, Dolly the sheep, was born.

“ CRISPR is a family of DNA sequences in bacteria and archaea. The sequences contain snippets of
DNA from viruses that have attacked the prokaryote. These snippets are used by the prokaryote to
detect and destroy DNA from similar viruses during subsequent attacks. These sequences play a key
role in a prokaryotic defense system, and form the basis of a technology known as CRISPR/Cas9 that

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A compilation by: Racquel A. Villanueva, RMT, MPH

 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY
(Medical Laboratory Science)

effectively and specifically changes genes within organisms. CRISPR is an abbreviation of Clustered
Regularly Interspaced Short Palindromic Repeats. The name was minted at a time when the origin
and use of the interspacing subsequences were not known. At that time the CRISPRs were described
as segments of prokaryoticDNA containing short, repetitive base sequences. In a palindromic repeat,
the sequence of nucleotides is the same in both directions. Each repetition is followed by short
segments of spacer DNA from previous exposures to foreign DNA (e.g., a virus or plasmid). Small
clusters of cas (CRISPR-associated system) genes are located next to CRISPR sequences. The
CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic
elements such as those present within plasmids and phages that provides a form of acquired
immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and
cut exogenous DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPRs are found in
approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea”.

GENETIC DIAGNOSIS AND SCREENING

The 2 ways by which DNA can be analyzed are by:

1. Direct DNA testing (direct examination of the genes) - is fast and sensitive but it can only be used if
the defective gene is known. E.g Mutation analysis for cystic fibrosis.

2. Indirect DNA testing (used of markers or linkage analysis) - is used if the type of gene mutation
that causes the disease is not yet idenfied. E.g prenatal diagnosis.

Polymerase Chain Reaction (PCR) can be used in the diagnosis of genetic diseases by providing
linkage analysis. It is an in vitro enzymatic method used to amplify DNA sequences. After DNA
amplication, mutations, causing the disorder can be detected using other diagnostic methods such as
endonuclease digestion, hybridization of the PCR product to an allele-specific oligonucleotide probe,
electrophoresis, DNA sequencing, and polymorphism analysis.

The PCR process: (Videos)

1. Denaturation- Breaking double-stranded DNA into single strands

2. Annealing- binding of a primer to DNA strand

3. Extension - elongation of DNA strand.

Ligase Chain Reaction (LCR) is an alternative procedure to PCR assay. It is used in the detection of
point mutations in inherited diseases.

Antisense Technology - this method inhibits a gene function by complementary base pairing to the
genetic target.

Transgenetic Technology - this method paves the way for introducing foreign genes into
experimental animals to make them transgenic. Transgenic animals provide a unique experimental
system for studying the function and regulation of genes of metabolic importance.

Major steps:

1. Microinjection of DNA into the pronucleus of single cell embryos

2. Retroviral infection of embryos or adult animals

3. Introduction of specific genes into embryonic stem cells, targeting them for homologous
recombination
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A compilation by: Racquel A. Villanueva, RMT, MPH

 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY
(Medical Laboratory Science)

4. Direct injection or carrier - mediated introduction of DNA into tissues of adult animals.

Implications of Genetic Testing

1. Genetic Discrimination. Results of genetic testing are part of an individual’s medical record.
Those who are at risk for certain genetic disorders can be denied life and/or health insurance. Being
stigmatized in school and the workplace is another form of genetic discrimination.

2. Eugenics. Negative eugenics is the belief that the human population can be improved by
preventing the reproduction of individuals with undesirable traits such as hereditary diseases.

3. Genetics Determinism. This is the belief that the person’s behavior, physical characteristics, and
personality are determined by his or her genes. Genetic determinism may result in faulty
generalization. For example, Africans are more susceptible to sickle cell anemia but their genes do
not indicate that they are predisposed to criminal behavior.

PRENATAL DIAGNOSIS

Prenatal screening involves testing for diseases or conditions of a fetus. The test done on in vitro-
fertilized embryos to detect genetic disorders in known as preimplantation genetic diagnosis. The
main purpose of prenatal screening is to detect birth defects such as Down’s syndrome, genetic
diseases, spina bifida, cleft palate, Tay Sachs disease, sickle cell anemia. Common testing procedures
and techniques include amniocentesis, sonograms, nuchal translucency, ultrasound, serum marker
testing and fetal screening.

Physical and socioeconomic factors can increase the risk of developing health problems and poor
birth outcomes. The following are some risk factors:

1. Age - females over the age 35

2. Weight - obese females

3. Previous pregnancy problems - such as having a premature baby or a baby with a birth defect.

4. Race- ethinic background susceptible to certain genetic diseases.

5. Family background- family history of a certain hereditary diseases.

6. Health condition- females with highblood pressure, diabetes, asthma, epilepsy, lupus, and history
of alcohol and drug abuse.

IN- VITRO FERTILIZATION

In-vitro fertilization is a procedures in which the union of a female’s egg and a male’s sperm take
place outside the body in an environmentally controlled chamber. The developed embryo is then
transferred to the woman’s uterus.

The basic steps involved in IVF are as follows: (Videos)

1. Stimulation. This is also known as superovulation because multiple eggs are stimulated and
brought to maturation and ovulation in this process. A fertitily drug is injected to stimulate the
female’s ovaries to release mature eggs. This drugs contains gonadotropins, which are naturally
produced by the female’s body. The two gonadotropins that stimulate the production of eggs are the
follicle stimulating hormone (FSH) and Luteinizing hormone (LH).

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A compilation by: Racquel A. Villanueva, RMT, MPH

 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY
(Medical Laboratory Science)

2. Follicular Aspiration. This is a procedure that involves the retrieval of eggs from the follicles
inside the ovary.

3. Insemination and Fertilization. The insemination process involves the mixing of the males’s
sperm with the female’s eggs ( collected using follicular aspiration). Fertilization can be done in two
ways: allowing the sperm to naturally enter the egg, or injecting the sperm into the egg in a process
known as intracytoplasmic sperm injection.

4. Embryo Culture. The fertilized egg divides and becomes an embryo. Embryo culture is a process
wherein the embryo is allowed to grow on an artificial medium. In this stage, preimplantation genetic
diagnosis maybe conducted for high-risk couples.

5. Embryo Transfer. The resultant embryo is transferred to female’s uterus through a thin tube.

Reasons for performing IVF

The main reason for a couple choosing IVF is infertility. Infertility in females can be caused by
advanced maternal age, endometriosis, and poorly functioning fallopian tubes. Infertility in males is
usually caused by decreased sperm count and the blockage of sperm transport. It is also an option for
impotent males suffering from erectile dysfunction or premature ejaculation.

Risk associated with IVF

1. Psychological stress and depression.

- bloating
- abdominal pain
- mood swings
- headaches
- ovarian hyperstimulation syndrome
- other side effects of fertility drugs.

2. High probability of giving birth to premature babies.

3. Higher probability of multiple pregnancy

4. Ovarian hyperstimulation syndrome.

SPERM BANKING

A sperm bank is a facility or a place where sperm is stored and kept viable under controlled
conditions. The sperm donor undergoes preliminary evaluation prior to donation. His seminal fluid is
then analyzed for volume, pH, sperm count, motility, progression, viability, abnormality, and white
blood cells. The sperms in the seminal fluid are mixed with medium before storing at low
temperature ( cryopreservation).

The sperms stored in such a facility can be used for the number of purposes:

1. Artificial Insemination. This is the technique that involves the injection of the sperms ( from the
sperm bank) into the female’s vagina or cervix. This is done during the ovulation period of the female.
The sperms travel to the fallopian tubes where it will fertilize the egg cells. Intrauterine insemination,
wherein the sperms are injected directly into the female’s uterus, is the most common form.

Gov. M. Cuenco Avenue, Banilad, Cebu City, Philippines 6000 * Tel # 032-342-0613 * Email Address: medtech@uc.edu.ph

A compilation by: Racquel A. Villanueva, RMT, MPH

 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY
(Medical Laboratory Science)

2. Surrogacy Arrangements and creating embryos for Embryo Donation.

3. Research or Educational Purposes.

4. Personal purposes.

SEX SELECTION AND PREDICTION

In sex selection and prediction, one sex is preferred over another; those individuals of the
nonpreferred sex would be at a disadvantage. This procedure would articially lead to an equal ratio
of females and males, and subsequently lead to discrimination, potential violence, and the abuse of
the smaller group. Sex selection can be considered as unnecessary tampering with nature, especially
among those who subscribe to the principles of law and ethics. On the other hand, it can control the
passing down of sex-linked and sex-influenced diseases, including hemophilia and parkinson’s
disease.

Stages:

1. Prefertilization. This method involves the separation of X-chomosomes-bearing and Y-

chromosomes-bearing sperms.

2. Postfertilization. PCR is used to determine the sex of the embryo by detecting the presence or
absence of a region of the sex-determining region Y (srY) in the embryo. The presence of the srY
protein means that the embryo will develop into a male.

3. Postimplantation. Techniques to determine sex during the postimplantation stage include:

- Ultrasound. Involves the use of high-frequency sound waves; this can be done during the 16 th
week of pregnancy.

- Chronic Villus Sampling (CVS). is done by obtaining placental tissue sample either through the
transvaginal or tansabdominal methods.

- Amniocentesis is a procedures where aminiotic fluid is collected , after which chromosomal

analysis is done on the amniotic fluid.

Karyotyping or chromosomal analysis is a test that evaluates the nu,ber and structure of the
chromosomes. The combination of X and Y chromosomes indicates a male fetus while the
combination of two X chromosomes indicates a females fetus.

ORGAN TRANSPLANTS

An organ transplant is the process of removing a failing or damaged organ in the body of the
recipient and replacing it with an organ from a donor.

Kidney Transplant. Nephrectomy is the surgical removal of kidney and can be carried out in to two
ways:

1. Laparoscopic nephrectomy. In this procedure, the surgeon makes 2 or 3 small incisions close to
the belly button. The kidney is removed through the central incision. Through one of the other
openings, a special camera called a laparoscope is used to produce an internal view of the abdominal

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A compilation by: Racquel A. Villanueva, RMT, MPH

 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY
(Medical Laboratory Science)

cavity. Surgeons use the laparoscope, which transmits a real-life picture of the internal organs to a
video monitor, to guide them through the surgical procedures.

2. Open nephrectomy. This is the traditional or conventional method of nephrectomy wherein the
kidney is removed via single large incision.

Liver Transplant. Liver transplant or hepatic transplant is a surgical procedure wherein the
damaged liver of the recipient is replaced by a portion or an entire liver from the donor. The process
wherein only a portion (allograft) is given to the recipient is known allotransplantation.

Lung Transplant. Lung transplant or pulmonary transplant is a surgical procedure that involves the
replacement of damaged lungs with the healthy lungs from the donor.

Types of lung transplants:

1. Lobe transplant - there are 2 donors; one will give a lobe from the left lung and the other will
donate a lobe from the right lung.

2. Single transplant - involves the donation of a single.

3. Double transplant - involves replacement of both lungs.

DNA CLONING

The process of creating a copies, or clones, of a single gene or DNA segment is known as DNA cloning.
The traditional method of DNA cloning or molecular cloning involves replication of DNA within a
living organism (in vivo). In this method, a fragment of DNA is introduced into a host cell and allowed
to replicate and produce exact copies of DNA. On the other hand, the in vitro method of creating a
copies of DNA does need a host cell. This is done by using polymerase chain reaction, which is a
laboratory technique for “amplifying” a specific DNA sequence. In DNA amplication, multiple identical
copies of DNA sequence are created.

DNA cloning is used in genetic fingerprinting; in genetic engineering to create plants with better
nutritional value or greater resistance to diseases and animals with desirable genetic features; in
protein production; and in sequencing genomes to decipher encoded protein protein or ribonucleic
acid sequences and protein expression.

Steps in DNA cloning using a vector.

1. Obtaining DNA fragments. Isolation of the desired DNA can be done by restriction-endonuclease
digestion, mechanical shearing, duplex cDNA synthesis, or direct chemical synthesis.

2. Joining the vector. The isolated DNA fragments can be transferred into a living cell through the use
of a cloning vehicle known as a vector. Joining is accomplished by homopolymeric tailing, ligation of
cohesive terminus, and the use of linker molecules.

3. Introduction to host cell. This step is called transformation because the function of the host cell
may be altered due to the fusion of the foriegn DNA with the native DNA. Transformation can be done
through the use of chemical poration or electroporation. Calcium chloride solution is used in the
chemical method because it increases the permeability of the cell membrane. In electroporation,
temporary pores are formed by exposing the cell to an electric pulse.

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A compilation by: Racquel A. Villanueva, RMT, MPH

 UNIVERSITY OF CEBU-BANILAD
COLLEGE OF MEDICAL TECHNOLOGY
(Medical Laboratory Science)

4. Selection. This steps involves determining which host cells have successfully replicated the desired
genes. Selection can be carried out using genetic or immunochemical methods, or nucleic acid
hybridization.

5. Expression. This involves the genetic expression of the received DNA. The information from the
received DNA is used in the synthesis of the desired genes.

References:
Suba, S.C. Et al (2013). Introduction to Medical Technology with Science, Technology and Society.

Jackson, R.A., Gibson, K.A., Wu, Y.W and Croughan, M.S. 2004. Perinatal outcomes in singletons
following in vitro fertilization: A meta-analysis. Obstrics & Gynecology.

http://pennstatehershey.adam.com/content.aspx?productId=117&pid=1&gid=007279>.

Ms Racquel A. Villanueva,RMT, MPH

Subject Head

Gov. M. Cuenco Avenue, Banilad, Cebu City, Philippines 6000 * Tel # 032-342-0613 * Email Address: medtech@uc.edu.ph

A compilation by: Racquel A. Villanueva, RMT, MPH