Download Restriction fragment length polymorphism

Tools and technique of molecular biology
Polymerase chain reaction (PCR)
• The polymerase chain reaction is an extremely
versatile technique for copying DNA.
• PCR allows a single DNA sequence to be copied
(millions of times), or altered in predetermined ways.
• PCR has many variations, like reverse transcription
PCR (RT-PCR) for amplification of RNA, and real-time
PCR (QPCR) which allow for quantitative
measurement of DNA or RNA molecules.
PCR Analysis
The process follows the principle of DNA replication
PRIMER
• A primer is a strand of nucleic acid that serves as a starting point
for DNA synthesis.
• These primers are usually short, chemically synthesized
oligonucleotides, with a length of about twenty bases. They are
hybredized to a target DNA, which is then copied by the
polymerase.
• minimum primer length used in most applications is 18
nucleotides.
• Replication starts at the 3'-end of the primer, and copies the
opposite strand.
• In most cases of natural DNA replication, the primer for DNA
synthesis and replication is a short strand of RNA .
How to see genes ...
... where in
tissues?
How to see specific
forms of genes?
How to clone
and amplify
genes?
southern blot
recombinant DNA
... where on
chromosomes?
in situ hybridization
genomic libraries
RFLP
How to analyze specific form of genes in genomic DNA?
Southern blotting
• Southern blot is a method for probing for the presence of a specific
DNA sequence within a DNA sample.
• DNA samples are separated by gel electrophoresis and then
transferred to a membrane by blotting via capillary action.
• The membrane is then exposed to a labeled DNA probe that has a
complement base sequence to the sequence on the DNA of
interest.
• less commonly used due to the capacity of other techniques, such
as PCR.
• Southern blotting are still used for some applications such as
measuring transgene copy number in transgenic mice, or in the
engineering of gene knockout embryonic stem cell lines.
Flow chart of Southern hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
MOLECULAR BIOLOGY – Molecular biology techniques
SOUTHERN BLOT
combines DNA fragmentation,
gel electrophoresis and hybridization
to analyze specific DNA sequences
Northern blotting
• The northern blot is used to study the expression patterns of a specific
type of RNA molecule as relative comparison among a set of different
samples of RNA.
• RNA is separated based on size and is then transferred to a membrane
then probed with a labeled complement of a sequence of interest.
• The results may be visualized through a variety of ways depending on the
label used. Most result in the revelation of bands representing the sizes of
the RNA detected in sample.
• The intensity of these bands is related to the amount of the target RNA in
the samples analyzed.
• It is used to study when and how much gene expression is occurring by
measuring how much of that RNA is present in different samples.
• one of the most basic tools for determining at what time, and under what
conditions, certain genes are expressed in living tissues.
Western blotting, or immunoblotting
Technique for detecting specific proteins separated by electrophoresis by use
of labeled antibodies.
Flow chart of Western blotting
Electrophoresing the protein sample
Assembling the Western blot sandwich
Transferring proteins from gel to nitrocellulose paper
Staining of transferred proteins
Blocking nonspecific antibody sites on the nitrocellulose paper
Probing electroblotted proteins with primary antibody
Washing away nonspecifically bound primary antibody
Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate
and formation of a diaminobenzidine (DAB) precipitate
Photographing the immunoblot
SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
Comparison of Southern, Northern, and Western blotting
techniques
Molecule
detected
Gel
electrophoresis
Gel
pretreatment
Southern blotting
DNA (ds)
Northern blotting
mRNA (ss)
Western blotting
Protein
Agarose gel
Formaldehyde
agarose gel
-
Polyacrylamide gel
Capillary transfer
cDNA, cRNA
Radioactive or
nonradioactive
Autoradiography
Chemiluminescent
Colorimetric
Electric transfer
primary antibody
Depurination,
denaturation, and
neutralization
Blotting method Capillary transfer
Probes
DNA
Radioactive or
nonradioactive
Detection
Autoradiography
system
Chemiluminescent
Colorimetric
-
Chemiluminescent
Colorimetric
GENOMIC LIBRARY
Hybridization
to filter
isolate
DNA
cloning
Molecular Diagnosis
Nucleic Acid Techniques:
• Plasmid profiling
(when characteristic
plasmids are available)
• RFLP analysis
(restriction fragment
length polymorphisms
- use RE to produce
characteristic gel
fragments)
• PCR (amplify key
sequences which
distinguish target
microorganisms)
Restriction Enzyme
Plasmid Analysis
Pattern
• plasmids are small, self• Cut DNA at specific
replicating circular DNA
location using
molecules found in many
natural enzymes:
bacteria
restriction
• plasmids often code for
endonucleases
resistance to antibiotics and
• get characteristic
certain virulence factors
fragments: “RFLP's /
• widely-used for tracking
restriction fragment
resistance in disease
length
outbreaks / pandemics
polymorphisms”
• can track transfer of
• see fragments via
resistance: between
electrophoresis
hospitals, organisms, and
• very accurate – can
countries
ID between microbial
• weakness - can transfer
strains
between microbes
Uses of Restriction Enzyme Patterns:
• identification of bacterial populations
• epidemiology (spread of disease
through population), pandemic science
• study of Tuberculosis in HIV-positive
patients
• combined with DNA fingerprinting
(southern blot, etc.) it is a very powerful
tool
PCR: Polymerase Chain
Reaction
• Making copies of a DNA
sequence
• PCR is conducted in vitro
(beaker / test tube)
• 20 cycles of PCR can
allow for a 1,000,000 X
amplification of DNA
samples
• Typical PCR reactions use
small (ng-mg) quantities
of DNA and go through
30-40 amplification
cycles
• PCR has revolutionized
R&D in biology /
medicine and helped
refine criminology and
law
Nucleic Acid Probes – MicroArray
•
•
•
•
•
can identify organisms at,
or below species level
can detect fastidious
organisms directly
(bacteria, viruses,
mycobacteria, fungi and
parasites)
Commercial kits available:
Gen-probe, Microprobe,
Digene etc. (all FDA
approved)
procedures are wellstandardized
use short, synthetic DNA
probes for well
understood characteristic
sequences
Gene Therapy
• Techniques that aim to cure an inherited
disease by providing the patient with a correct
copy of the defective gene
• Can include “gene addition” or “gene
subtraction”
Gene Therapy for Inherited Diseases
• Germline therapy – uses a fertilized egg, so the gene is present in all cells
of the resulting individual
•
Somatic cell therapy - good for inherited blood diseases (e.g., haemophilia,
thalassemia; stem cells from the bone marrow) and lung diseases (e.g., cystic
fibrosis; periodic inhaling of DNA in rats)
•
no good method available yet for replacing a defective gene (necessary for a
dominant one)
•
Current therapies add a gene but can’t replace the defective information yet
Gene Therapy and Cancer
• gene therapy can be
applied not only to
inherited / infectious
diseases but also cancer
• specific killing of cancer
cells using cancerspecific promoters and
toxin genes
• cause tumor cells to
synthesize
strong
antigens
that
are
efficiently recognized by
the immune system
• suitable
delivery
methods
to
the
cancerous cells are not
yet available
4. Stem Cells
Stem cells are poised to
revolutionize
medical
science:
• Re-grow
damaged
tissues
• Fix otherwise lethal
abnormalities
• Potential to repair birth
defects
before
symptoms ever appear
• Cure
“incurable
diseases” (MS, ALS, etc.)
Somatic Stem Cells
• Somatic stem cells occur in
different types:
• Haematopoietic stem cells –
blood-forming stem cells are
found in the bone marrow as well
as the umbilical cord of newborn
babies.
• Stromal stem cells – (bone
marrow cells) can differentiate
into cartilage, fat/adipocytes and
bone.
• Neural
stem
cells
–can
differentiate into various neural
cells including neurons and the
myelin-sheath
producing
oligodendrocytes.
Embryonic Stem Cells
• Derived from cells of the inner
cell mass of the blastocyst (<5
day old embryonic cell mass) typically has less than 160 cells
in total.
• Like Somatics, Embryonic stem
cells
have
two
core
characteristics:
• an unlimited capacity to selfreplicate
•
the capability (potency) of
differentiating into any one of
the more than two hundred
identified tissue types found in
the human body.
Molecular markers
• Molecular marker are based on naturally occurring
polymorphism in DNA sequence(i.e. base pair deletion,
substitution ,addition or patterns).
• Genetic markers are sequences of DNA which have been
traced to specific locations on the chromosomes and
associated with particular traits.
• It can be described as a variation that can be observed.
• A genetic marker may be a short DNA sequence, such as
a sequence surrounding a single base-pair change (single
nucleotide polymorphism, SNP), or a long one, like mini
satellites.
Some commonly used types of genetic markers are
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RFLP (or Restriction fragment length polymorphism)
AFLP (or Amplified fragment length polymorphism)
RAPD (or Random amplification of polymorphic DNA)
VNTR (or Variable number tandem repeat)
Micro satellite polymorphism, SSR (or Simple sequence
repeat)
SNP (or Single nucleotide polymorphism)
STR (or Short tandem repeat)
SFP (or Single feature polymorphism)
DArT (or Diversity Arrays Technology)
RAD markers (or Restriction site associated DNA markers)
There are 5 conditions that characterize a
suitable molecular marker
• Must be polymorphic
• Co-dominant inheritance
• Randomly and frequently distributed
throughout the genome
• Easy and cheap to detect
• Reproducible
Molecular markers can be used for several
different applications including
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Germplasm characterization,
Genetic diagnostics,
Characterization of transformants,
Study of genome
Organization and phylogenic analysis.
Paternity testing and the investigation of crimes.
Measure the genomic response to selection in
livestock
RFLP (Restriction fragment length polymorphism)
RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which
can recognize and cut DNA wherever a specific short sequence occurs. A
RFLP occurs when the length of a detected fragment varies between
individuals and can be used in genetic analysis.
Advantages:
• Variant are co dominant
• Measure variation at the level of DNA sequence, not protein sequence.
Disadvantage:
• Requires relatively large amount of DNA
AFLP ( Amplified fragment length polymorphism)
In this analysis we can amplify restricted fragments and reduces the
complexity of material to be analyzed (approx 1000 folds).it can be
used for comparison b/w closely related species only.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either homozygous
or heterozygous for the Sequence - can’t tell which?
RAPD ( Random amplification of polymorphic DNA)
Random Amplification of Polymorphic DNA. It is a type of PCR
reaction, but the segments of DNA that are amplified are random.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either
homozygous or heterozygous for the Sequence - can’t tell which?
• Data analysis more complicated
Micro satellite polymorphism, SSR or Simple
sequence repeat
Microsatellites, Simple Sequence Repeats (SSRs), or Short
Tandem Repeats (STRs), are repeating sequences of 1-6 base
pairs of DNA.
Advantages:
• Highly variable
• Fast evolving
• Co dominant
Disadvantage:
• Relatively expensive and time consuming to develop
SNP
• A single-nucleotide polymorphism
(SNP, pronounced snip) is a DNA
sequence variation occurring when
a single nucleotide — A, T,C, or G
— in the genome (or other shared
sequence) differs between
members of a species or paired
chromosomes in an individual.
• Used in biomedical research ,crop
and livestock breeding programs.
STR
• A short tandem repeat (STR) in DNA occurs when a
pattern of two or more nucleotides are repeated and the
repeated sequences are directly adjacent to each other.
• The pattern can range in length from 2 to 16 base pairs
(bp) (for example (CATG)n in a genomic region) and is
typically in the non-coding intron region
• Used in forensic cases.
• used for the genetic fingerprinting of individuals