Molecular Technology

1. Molecular Biotechnology
2 & 3

2. Genomic DNA and complementary DNA (cDNA)
Two methods are commonly used to produce
target DNA pieces suitable for cloning: genomic
DNA and complementary DNA (cDNA).
1- Genomic DNA is digested with restriction
endonucleases producing a mixture of all or part of a
particular gene, including introns and other
noncoding sequences.
2- cDNA is produced from messenger RNA (mRNA)
isolated from a particular tissue, and it does not
contain introns.
Reverse transcriptase produces ssDNA strand
complementary to the mRNA. the resulting DNA-
mRNA hybrid is denatured, DNA polymerase is
used to produce double-strand cDNA from the single-

3. When to use cDNA or gDNA?

4. Probes
a Hybridization Probe is a Fragment of DNA or RNA of Variable Lengths
(usually 100–1000 Bases Long) which can be Labeled
Probes can be used in DNA or RNA Samples to Detect the Presence of
Nucleotide Sequences (the DNA Target) that are Complementary to the
Sequence in the Probe
Labeling & Detection of Probes:
• Radioactive Labeling: using 32P……detected by X Ray films
• Florescent Labelling: using Florescent Dye .detected by UVR
• Chemical Labeling: using Enzyme-Substrate to give Color detected by
Spectrophotometer

6. Blotting Techniques
Molecular Technique used for Detection of Specific DNA or RNA
Fragments or protein molecules among the Many Thousand
Molecules
Types:
1- Southern Blotting : used for Detection of Specific DNA in DNA
Samples
2- Northern Blotting : For Detection of a Specific mRNA in RNA
Samples.
3- Western Blotting : for detection of proteins

7. Southern Blotting Technique
1- Cleavage: DNA is extracted from cells then
cleaved into many fragments by restriction
enzyme(s)
2- Electrophoresis: The resulting DNA fragments
are separated on the basis of size by
electrophoresis. The larger DNA fragments move
more slowly than the smaller fragments
3- Blotting: DNA is denatured (to single strands i.e.
ssDNA). DNA is transferred from the gel to a nylon
membrane
4- Hybridization: Special radioactive labeled probes
having complementary bases to specific gene
searched for are applied in membrane where they
will combined with the gene
5- Autoradiography: x ray film To detect the
radioactive bands

8. Southern Blotting Technique Steps

10. Gene expression analysis
1- Determination of mRNA level
• Northern blots: mRNA separated by electrophoresis gel →
transferred to cellulose membrane → identified by labeled probe
• RT PCR, real time PCR
• Micro-arrays
2- Protein analysis:
– ELISA,
– RIA
– Western blots (more sensitive and specific)
3- Proteomic:
study all proteins expressed from genome for function, interaction,
post-translation modifications

11. Micro-arrays Ships
• It is a collection of microscopic DNA spots attached to a solid surface.
• Scientists use DNA microarrays to measure the expression levels of large
numbers (thousands) of genes simultaneously or to genotype multiple
regions of a genome.
 1st mRNA → cDNA by Reverse Transcriptase (RT)
 1000s of ssDNA fixed on a Chip (Complementary Sequence to Target cDNA →
bind with Target cDNA → laser beam → computer to detect the Target cDNA
 Quantify Level of mRNA Expression or Detect Mutation or Sequencing

13. Comparative gene expression study

14. • Genome of different people are 99.5% identical. Only 0.5% shows variation which
cause polymorphism (Many Forms) which occur mainly (98%) in DNA non coding
protein as introns, intergenic region
• DNA Polymorphism is any difference in the Nucleotide Sequence Among
individuals
• i.e. DNA Sequence Variation that is NOT associated with Any Observable
Phenotypic Variation & can Exist Anywhere in the Genome (Not necessarily in a
Gene).
• These Differences can be Single Base Pair Changes, Deletions, Insertions or even
changes in the number of copies of a given DNA sequence.
POLYMORPHISM

15. • Genetic variant that can be observed by
cleaving the DNA into fragments (Restriction
Fragments, RF) with a restriction enzyme.
• The Length of the Restriction Fragments (RFL)
is altered if the variant alters the DNA by
creating or abolishing a site of restriction
endonuclease cleavage.
• Restriction Fragment Length Polymorphism
(RFLP) can be used to detect human genetic
variations.
Two Types of DNA Variation Commonly result in
RFLP:
1- Single Nucleotide-Base changes (SNP)
RESTRICTION FRAGMENT LENGTH POLYMORPHISM ( RFLP)

16. 1- Single Nucleotide Polymorphisms (SNPs )
 SNP is Single Base Pair change, Point mutation & site is called SNP Locus. Constitute
about 90% of Human Genome Variation
 The substitution of one nucleotide at a restriction site can render the site
unrecognizable by a particular restriction endonuclease or/ a new restriction site
can also be created by the same mechanism
 In either case, cleavage with an endonuclease results in Fragments of Lengths
Differing from the normal that can be Detected by DNA Hybridization
DetectionofSickleCellAnemiabySNP

17. 2- Variable Number of Tandem Repeats (VNTR)
• VNTR are short sequences of DNA at scattered locations in the genome, repeated
in tandem (one after another).
• The number of these repeat units varies from person to person but is unique for any
given individual & therefore, serves as a molecular fingerprint.
• Cleavage by restriction enzymes yields fragments that vary in length depending on how
many repeated segments are contained in the fragment, Size determined by gel
electrophoresis & Southern blotting to produce pattern of bands unique to each
individual (so, it is essential to forensic crime investigation).

18. o
DNA Fingerprinting
DNA fingerprinting is a laboratory technique used to establish a link between biological
evidence and a suspect in a criminal investigation.
• The probability of having two people with the same DNA fingerprint is
very small.
• DNA fingerprint is something that someone is born with, it is unique to
him.
• One half of DNA is from the mother & one half from the father.
• DNA profiling is commonly used to provide evidence that someone is, or
is not, the biological parent of a child (paternity Testing or Forensic
Testing)

19. Transgenic animal
Transgenic animal is an animal that carry a foreign gene that has been inserted in its
genome in germ line stage (ova or sperm or zygote).
Methods to produce transgenic animal:
1- pronuclear microinjection
2- Embryonic stem cell method
3- Retrovirus mediated gene transfer

20. Applications of Transgenic animal :
1-Farmers have always used selective breeding to produce animals
that exhibit desired traits (e.g., increased milk production, high
growth rate ) milk with less lactose or cholesterol
2-To produce disease-resistant animals, such as influenza-
resistant pigs,
3- Nutritional supplements and pharmaceuticals
Products such as insulin, growth hormone, and blood
clotting factors may soon be or have already been obtained
from the milk of transgenic cows, sheep, or goats
4- Models for human diseases and treatments rigmens

21. Gene Therapy
• Gene therapy is a technique for correcting defective genes responsible for
disease
Types of Gene Therapy
• 1- somatic cells therapy (most cells of the body) All gene therapy has
been directed at somatic cells. delivered gene donot not pass to the next
generation
• 2- Germline therapy (such as sperm cells, ova, and their stem cell
precursors). For the introduced gene to be transmitted normally to
offspring as transgenic animals

22. Types of Vectors in Gene Therapy
1- Viral Gene Delivery:
A- Retroviruses (as HIV)
 A class of viruses that can create double-stranded DNA copies of their
RNA genomes
 These copies of its genome can be integrated into the chromosomes of
host cells
 Producing a DNA copy from an RNA molecule is reverse transcription
• New DNA must be incorporated into the genome of the host cell. This
process is done by another enzyme carried in the virus called integrase
Problems with Retroviruses
• If genetic material is inserted in the middle of
one of the original genes of the host cell, this
gene will be disrupted (insertional mutagenesis).
• If the inserted gene is one regulating cell
division, uncontrolled cell division can occur
(i.e. cancer)

24. B- Adenoviruses (as HIV)
• A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal
and eye infections in human
• Genetic material of adenoviruses is not incorporated into the host cell's genetic
material. So, DNA is left free in the nucleus of the host cell & the instructions in this
extra DNA molecule are transcribed just like any other gene.
• Absence of integration into the host cell's genome should prevent the possibility of the
occurrence of cancer.
Problems with Adenovirus:
• The extra genes are not replicated. So, the descendants of that
cell will not have the extra gene. As a result, treatment with the
adenovirus will require re-administration in a growing cell
population.

25. 2- Non viral Gene Delivery
1. Direct Introduction: Naked DNA
introduction of therapeutic DNA into target cells. This approach is limited in its application
because it can be used only with certain tissues and requires large amounts of DNA
2. Liposome: Coated DNA
Creation of an artificial lipid sphere with an aqueous core. This carries the therapeutic DNA,
is capable of passing the DNA through the target cell's membrane.
3. Chemical Linking: Linked DNA
Chemically linking the DNA to a molecule that will bind to special cell receptors. Once
bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane
and passed into the interior of the target cell.

26. What factors have kept gene therapy from becoming an effective treatment
for genetic disease?
1. Problems with integrating therapeutic DNA into the genome and the
rapidly dividing nature of many cells prevent gene therapy from achieving any
long-term benefits Patients will have to undergo multiple rounds of gene
therapy.
2. Immune response: a foreign object is introduced into human tissues, the
immune system is designed to attack the invader.
3. Problems with viral vectors: Viruses carrier present a variety of potential
problems to the patient --toxicity, immune and inflammatory responses, In
addition, there is always the fear that the viral vector, once inside the patient,
may recover its ability to cause disease.
4. Multigene disorders? Conditions or disorders that arise from mutations in a
single gene are the best candidates for gene therapy.

27. Genetic counseling
Genetic counselling is the utilization of knowledge of genetics to predict the probability of
genetic disorders.
It is of 2 types:
1- Prospective genetic counselling: before marriage
- In this case, the genetic disorder has not yet expressed itself
- It is done for heterozygotic individuals to assess the probability of having a child with
genetic disorders
- If a person is identified as heterozygotic for a genetic condition, he/she should be
advised against marrying another heterozygotic individual as there is increased risk of
the trait expressing itself in the phenotype
2- Retrospective genetic counselling: after marriage
- In this case, the disease has already occurred in the family
- It is more commonly practiced as compared to prospective genetic counseling as
people usually come for genetic counseling only after having a child with congenital
anomalies / mental retardation / inborn errors of metabolism
- The interventions as a part of retrospective genetic counselling are:
• Contraception
• Sterilization

28. Making Sense of Your Genes: A Guide to Genetic Counseling
How can I prepare for a genetic counseling visit?
• Come to the visit with a list of questions you would like to ask. This
will help the counselor focus on your concerns.
• Genetic counseling visits usually involve collecting family history
information. It can be useful to ask your relatives about what types of
medical conditions occur in your family before your visit.
• If you have medical records relating to your concerns, you may want
to bring them or ask your doctor to send them to the genetic
counselor before your visit.

30. What can you expect from your visit?
Common Topics include:
• Talking about your family health history & ethnic heritage.
• Helping you understand the causes of genetic conditions.
• Helping you understand testing options, diagnosis or in some cases, the reason why no
diagnosis has been made.
• Guiding you through decision-making about genetic testing, family planning, or medical
planning.
• Helping you deal with emotions associated with having or not having a known genetic
condition, having a relative with a genetic condition or being at risk for a genetic
condition.
• Finding supportive resources to help you manage a genetic condition.
• Understanding the chance of passing a genetic condition on to your children.
• Your input is very important to the genetic counseling session; the details you provide
will allow the genetic counselor to fully understand your health concerns. A genetic
counseling session is a conversation. Your input is very important to the session.

31. Questions you might ask your genetic counselor
• Does the disease in question run in families?
• If my family member has a disease, might I get it?
• If I have a disease, are my family members at risk of getting it?
• Is any kind of genetic testing available? If so, what are the benefits and limitations of
the testing? How will I pay for it?
• What kind of information can genetic testing give me?
• What does the genetic testing process involve?
• If I decide to have genetic testing for myself or my child, when can I expect to hear
about the results? Will the results be given to me over the phone or in person?
• How can knowing more about a genetic risk help me?
• Could I be exposing myself or my family to discrimination based on genetic
information?