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Chapter 3 Part 2

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5pn4x4czxn
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CHAPTER III

MOLECULAR TECHNIQUES
MAE ROSE M. MAOIRAT-ABAD
CHAPTER OUTLINE

 DNA Extraction
 Agarose Gel Electrophoresis
 Hybridization
 Polymerase Chain Reaction
 DNA sequencing
 Bioinformatics
HYBRIDIZATION

 process in which two single-stranded nucleic acid (NA) molecules with


complementary base sequences form a double-stranded NA molecule
 used for detection of gene and gene expression, determining the location of genes,
diagnosis of diseases
HYBRIDIZATION

Principles of Hybridization
 Hybridization technique was developed on the basis of denaturation and renaturation
of nucleic acids.
HYBRIDIZATION
 Nucleic acid hybridization as a technique involves using a
labeled nucleic acid probe which is a known DNA or RNA
fragment, to bind with the target nucleic acids, which is
usually a poorly understood, heterogenous population of
nucleic acids.
 A probe labeled with detectable tracer is the prerequisite for
determining a specific DNA sequence or gene in a sample or
genomic DNA by nucleic acid hybridization
 The target nucleic acids to be analyzed are usually denatured,
and then mixed with the labeled probe in the hybridization
system.
 The hybridization can be identified by the detection of the
tracer labeling the probe. Thus the existence or the
expression of a specific gene can be determined.
PREPARATION AND LABELING OF NUCLEIC ACID

Probe preparation
 Probes must be single stranded molecule
 The probes used in hybridization includes oligonucleotides (15-50 nucleotides),
genomic DNA fragment, cDNA fragment, and RNA
PREPARATION AND LABELING OF NUCLEIC ACID

Oligonucleotide probes
 Are short fragments designed with a specific sequence complementary to the given
region of the target DNA.
 Usually synthesized in vitro

Genomic DNA probes


 Can be prepared from the cloned DNA fragment in plasmid
PREPARATION AND LABELING OF NUCLEIC ACID

cDNA probes
 Can be prepared from the cloned cDNA in plasmid, or amplified directly from mRNA by
RT-PCR

RNA probes
 Are usually transcribed in vitro from a cloned cDNA in a proper vector.

The size of genomic DNA probes, cDNA probes, and RNA probes may be 0.1kb to
1kb
PREPARATION AND LABELING OF NUCLEIC ACID

Labeling of probes
 Probe is usually labeled with a detectable tracer, which is either isotopic or non-
isotopic. The purified oligonucleotide is labeled in vitro by using a suitable enzyme to
add the labeled nucleotide to the end of the oligonucleotide
 For the preparation of the labeled RNA probes, RNA probes are usually synthesized
by RNA polymerase in the presence of ATP, GTP, CTP and the UTP, with specific
fragment of a gene or cDNA in a proper vector as template. RNA probes can then
be generated and be labeled at the same time.
PREPARATION AND LABELING OF NUCLEIC ACID

Labeling of probes
 Genomic DNA probes and cDNA probes are usually labelled in the process of DNA
synthesis in vitro. In the reaction of DNA synthesis with a DNA probe as template, if
a labeled-dNTP, which can be incorporated into newly synthesized DNA chain, is
added as a substrate, the labeled DNA probe will be formed.
 The labels commonly used include radioactive (32P and 35S) and non-radioactive
(digoxigenin, biotin, fluorescein) substances
TYPES OF HYBRIDIZATION

SOUTHERN BLOT
 Assay for DNA-DNA hybridization
STEPS
1. Digest DNA with restriction enzymes or endonucleases
2. Load DNA fragments in the gel and apply electrical current (gel electrophoresis)
3. Stain the gel
4. Denature the double-stranded fragments by soaking the gel in alkali solution
5. Transfer the DNA to a membrane
POLYMERASE CHAIN REACTION (PCR)
POLYMERASE CHAIN REACTION (PCR)

 A technique for amplifying DNA sequences


in vitro by separating the DNA into two
strands and incubating with oligonucleotide
primers and DNA polymerase
 It can amplify a specific sequence of DNA as
many as one billion times
PCR REQUIREMENTS

 DNA template
 Oligonucleotide primers
 Taq polymerase
 Deoxynucleotide (dNTPs)
 Buffer solution
 Divalent cations (Mg2+)
 Thermal cycler (PCR machine)
STEPS INVOLVED IN PCR

PCR is repeated cycle for amplifying target DNA. Each cycle has three stages

I. Denaturation of double stranded DNA template


II. Annealing of primers
III. Extension of double stranded DNA molecule
STEPS INVOLVED IN PCR

DENATURATION
 Temperature: 92-96C
 Time: 1 min
 DNA is denatured
STEPS INVOLVED IN PCR

ANNEALING
 Temperature: ~50-70C
 Time: 1.5 min
 Primers bind to the complementary
sequences
STEPS INVOLVED IN PCR
EXTENSION
 Temperature: ~72C
 Time: 0.5-1 min
 DNA polymerase bind to the annealed primers and extends DNA at the 3’ end of the
chain
 Each cycle of PCR takes about 3-5 minutes
DNA SEQUENCING
DNA SEQUENCING

 A process of determining the precise order of nucleotide within the DNA molecule
 The first DNA sequencing was done in 1975 by Frederick Sanger
 This method is called Sanger Sequencing
 It used the termination method
SANGER SEQUENCING

 Sanger method uses non-real, radioactive-labeled bases that help


stop DNA replication
 These fake nucleotides are colored corresponding to the real
nucleotides
 First step is to create multiple copies of the DNA through PCR
 Fragments are heated again to make single stranded DNA
 Primer and dNTPs are added
 The DNA is run in gel electrophoresis

https://www.youtube.com/watch?v=KTstRrDTmWI
USES OF DNA SEQUENCING

 Used in the field of functional genomics, to understand which DNA codes for which
gene, and codes for what protein
 Used in comparative genomics to understand the organisms history
 Improve health care
 Help animals and plants to resist certain diseases
 In forensic science, to identify bodies and criminals
SEQUENCING TECHNOLOGIES

1st generation sequencing Next Generation Sequencing (NGS)


 Sanger Sequencing -high throughput sequencing
 Maxam-Gilbert Sequencing - Sequence thousands at once
- Massive parallel sequencing
2nd generation  Sequencing by synthesis
 Ilumina MiSeq  Sequencing by ligation
 Genereader  Pyrosequencing
 Pyrosequencer  Ion semiconductor
BIOINFORMATICS
BIOINFORMATICS

 It is the application of information technology to store, organize, and analyze the vast
amount of biological data
 The data is in the form of sequences of DNA and amino acids, and structures of
proteins
 Sequences are represented in single dimension, whereas structure contains the three
dimensional data
BIOINFORMATICS

 It is the merging of biology, mathematics, statistics, computer science, information


technology, chemistry into a single discipline to process biological data
 Needs complex machines to read and analyze biological data (super computers)
 The term “Bioinformatics” was coined by Paulien Hogeweg and Ben Hesper in 1970
IMPORTANCE OF BIOINFORMATICS

 Gain a better understanding of gene analysis, taxonomy, and evolution


 To work efficiently on drug designs and reduce time taken for the development of
drug manually.
 Used in the area of molecular medicine
 For environment, in the identification of waste and clean-up bacteria
 In agriculture, to produce high yield and low maintenance crops; drug discovery for
animals
APPLICATIONS

1. Prediction of protein structure – proper protein function


2. Genome annotation – gene identification
3. Comparative genomics – for evolution
4. Drug discovery
5. Preventive medicine
6. Gene therapy
SOFTWARE AND TOOLS

 BLAST
 Expasy
 UniProt/SwissProt
 Entrez
 NCBI
 EMBL-EBI
 ENSEMBL

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