DNA TECHNOLOGIES

Anamika Sengupta PhD, MEd

Asst. Professor
Biochemistry/Academic Success, MERP
asengupta@devrymedical.org
 Learning Objectives
1. Define RE; summarize their role in DNA technology; Define & characterize
recognition sequences; differentiate between sticky/blunt ends

2. Define recombinant DNAs; Reflect on the role of rDNAs in gene cloning; learn
the important of plasmids vectors

3. Focus on definition & importance of cDNA in molecular biology

4. Define hybridization probes. How does it differ from ASO probes; Understand
the role of ASO probes in detection of homozygosity/ heterozygosity of gene
mutation

5. Emphasize of procedural details and the clinical importance of electrophoresis,

different blotting techniques; understand Sanger method for nucleic acid
sequencing

6. Learn the underlying principle, procedural details and clinical importance of

polymerase chain reaction (PCR). Emphasize on the role of primers in PCR.
7. Acquire a basic understanding of gene therapy
 1
History
Before 1970, DNA was the most difficult
cellular molecule to analyze and work with
for biochemists 2

In 1973, 3 major technological

breakthroughs in genetic engineering were

1. Discovery of a Restriction Endonuclease

(DNA scissors)
3
2. Invention of DNA cloning

3. Construction and use of DNA probe

 Restriction Endoucleases (RE)
Definition: Bacterial enzymes capable of cutting a DNA molecule
at strictly defined sites called recognition sites producing
double stranded fragments of average sizes.

Commonly used RE: EcoR1, Hpa1, Hind III, Pst1

 Recognition Sites

4-8 nbp long double stranded palindromes

called recognition sites

May be different/same for different RE.

Recognition site: 4 nbp
May be ambiguous or unambiguous

Length often dictates the frequency of cuts Fragment length: 256nbp

produced
Cleavage distance:256 nbp
 Patterns of DNA cuts by RE
Commonly used RE cut within the recognition sites and generate
one of the 3 types of ends

5' overhangs:

3 overhangs:

The 5' / 3' overhangs generated by asymmetric cuts are called

sticky ends or cohesive.
Blunt ends: The two strands of DNA symmetrically at precisely
opposite sites,

Not popular in rDNA technology

Joining 2 fragments with blunt ends gives low yield

2 DNA fragments with cohesive/sticky ends from 2 sources can

be joined easily if they are cut using the same RE
USE of RE: Creating Recombinant DNA (rDNA)
Requires:
Two DNA fragments from different
sources

Both fragments with sticky ends

produced by the same restriction
endonuclease

DNA ligase: for joining the 2 DNA

strands

Product is a recombinant DNA

(rDNA)
 Use of Recombinant DNA: DNA Cloning
What is DNA cloning?
Producing multiple copies of a single gene or DNA fragment

Steps of DNA cloning

Isolation of the DNA of interest
Purification and fragmentation
Insertion into a vector
Incorporation of the vector into bacterial host cells (E.coli), a
process called transformation
Replication of E. coli producing multiple copies of each DNA
fragment
Summary of Gene Cloning
Vectors used in Recombinant DNA Technology
Definition: An agent that help to transfer and replicate a foreign DNA
within a specific host cell (E.coli)

Major types: Plasmids (commonly used), viruses

Structural features :

Ori (origin of replication) site: High copy numbers of plasmid/E.coli cell

Antibiotic resistant gene: Helps in selection

Recognition sites: For cleavage by RE

Insertion site for the transgene

To target the coding regions (exons)

a. mRNA is purified from the target

cell

b. It is reverse transcribe using reverse

transcriptase enzyme

c. Product: Single stranded DNA

complementary DNA (cDNA)
 Hybridization Probes
Single or double stranded fragments of DNA/RNA (100-1000
nbp)

Tagged to a radioactive/fluorescent/enzyme marker

Helps to detect a target DNA present in a complex nucleic

acid mixture

Detection Process: Complementary base pair hybridization

A probe is commonly generated by chemical synthesis

Detection of target DNA using a fluorescent probe

Electrophoresis

Blotting
 Electrophoresis and Blotting
Agarose gel electrophoresis: Separates
Electrophoresis DNA/RNA according to size
(Molecular technique that
separates complex DNA Polyacrylamide gel electrophoresis:
/RNA/protein mixtures) Separates proteins/small nucleic acids
according to size and net charge

Blotting
Nitrocellulose membranes: Preferred
Involves 2 steps:
membrane for protein blotting
1. Transfer of separated Nylon membranes: Preferred
DNA/RNA /Protein fragments
from the gel to a membrane
membrane for DNA/RNA blotting

2. Probing of separated Southern Blot: Probing of DNA

DNA/RNA/ Protein mixture for Northern Blot: Probing of RNA
a sequence of interest Western blot: Probing of proteins
Fig: Process of electrophoresis
 Steps of Southern Blotting

1.Digestion of DNA with RE

2.Separation of DNA fragments

by agarose gel electrophoresis

3.Denaturation of dsDNA
fragments into ssDNA

4.Blotting of DNA from the gel

onto a membrane

5.Detecetion of target DNA

with a labeled probe
 Allele Specific Oligonucleotide (ASO) Probes
15-20 nbp long synthetic DNA probes Detection of sickle cell
anemia
Used in pairs

Detects differences in nucleotide

sequences between 2 alleles of a gene
(i.e homozygosity/heterozygosity of a
gene mutation)

Example: Detection of Sickle cell

anemia
 Sickle Cell Anemia

What is sickle cell

anemia??

Sickle shaped RBC

 DNA Sequencing by Sanger Method
Requirements
ssDNA of unknown sequence (template)

Labeled DNA primers, DNA polymerase enzyme

Deoxynucleotide triphosphates (dNTPs): dATP, dCTP, dGTP, dTTP

Dideoxynucleotide triphosphates (ddNTPs): ddATP, ddCTP, ddGTP,

ddTTP or chain terminating nucleotides

Summary of the procedure

Products of the 4
reactions are
Split sample in 4 Synthesis of separated by gel
Add primers, tubes; add complementary electrophoresis in
Template DNA dNTPs, DNA different strand stops on 4 separate lanes;
polymerase ddNTPs to each incorporation of Gel is read from
tube ddNTPs the bottom to the
top across each
lane
 Why are ddNTPs called chain terminators?

ddNTPs lack a 3OH on the sugar

moiety

Extension of daughter strand stops

at the point of incorporation of the
ddNTP

Reason: Prevents formation of

phosphodiestor bond between the
incorporated ddNTP and the
incoming dNTP
 Diagrammatic Presentation of Sanger Method
3 TGCACTTGAACGCAT 5 Single stranded DNA

5 ACGT 3
Labeled Primer

+ DNA Polymerase
+ dATP, dTTP, dCTP, dGTP

+ ddATP + ddTTP + ddCTP + ddGTP

ACGTGAACTTGCGTA ACGTGAACTTGCGT ACGTGAACTTGC ACGTGAACTTGCG

ACGTGAA ACGTGAACTT ACGTGAAC ACGTGAACTTG

ACGTGA ACGTGAACT
Read sequence

Electrophoresis
5 GAACTTGCGTA 3

3 CTTGAACGCAT 5
Read gel
Complementary sequence
 Polymerase Chain Reaction (PCR)
Definition: Molecular technique; allows DNA to be effectively
amplified billion fold in a cell-free system (in vitro); Uses
temperature sensitive enzyme dependent reactions; requires
2-3 hr

Requirements

1. Template DNA 2. DNA primers

3. DNA polymerase enzyme 4. Nucleotides (dNTPs)

5. Adequate amount of buffers 6. Automated Thermal

cycler
Steps in PCR: 3 major temperature
dependent steps; 30-40 reaction
cycles

1. Denaturation (94- 95C for I min)

2. Annealing (55- 70C for 45 sec)

3. Extension (72C for 2 min)

Exponential Amplification of the Target Gene by
PCR
 End Point Analysis of the Amplified Product in
Conventional PCR
Verified by agarose gel electrophoresis
 PCR in Newly Emerging Laboratory & Clinical
Techniques

DNA Fingerprinting/DNA profiling

Detection of disease caused by blood pathogens

HIV
Hepatitis B, C
Malaria (detection of circulating Plasmodium falciparum DNA)

Blood screening

Genetic screening (detects predisposition of an individual to

conditions like cancer, heart diseases)
 Pre/Post-Implantation Genetic Diagnosis

Pre-implantation Prenatal DNA testing Non invasive

genetic diagnosis for chromosomal prenatal diagnosis
1. Thalassaemia abnormalities/ using circulating
monogenic disorders fetal DNA in
2. Cystic fibrosis maternal plasma
 Polymerase chain reaction (PCR) involves 3 basic temperature
dependent enzyme sensitive reactions that occur sequentially for 30-
40 cycles leading to exponential amplification of a gene of interest in a
cell free system. What is the proper order in which the 3 temperature
sensitive reactions occur during a PCR cycle?

A. Annealing, denaturation,
extension
B. Extension, annealing,
denaturation
C. Denaturation extension,
annealing
D. Denaturation, annealing,
extension 0% 0% 0% 0% 0%
E. Annealing, extension,
denaturation

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 Gene Therapy
What is gene therapy?
A technique for correcting defective genes that lead to disease
development

Approaches in gene therapy

Insertion of a normal gene at a non-specific locus (most common
approach)

Replacement of the abnormal gene with its normal copy by

homologus recombination

Repair of the mutated gene through selective reverse mutation

Altering the regulatory elements of a gene to rectify its function

Modes of Delivery of Therapeutic Genes

Direct or cell based

delivery
THANKS