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MOLECULAR BIOLOGY TECHNIQUES

Dr. Mohammed Shakil Akhtar

Lecture Outlines
Restriction Edonucleases.
- Introduction - Types - Nomenclature - Characteristics - Importance Gel Electrophoresis. DNA Fingerprinting. Recombinant DNA ( DNA cloning) Polymerase Chain Reaction (PCR). - Requirements of PCR - Steps of PCR - Applications of PCR. DNA sequencing.

Restriction Endonucleases (RE)


Restriction endonucleases (restriction enzymes) cleave
double-stranded DNA into smaller, more manageable fragments.

Each restriction enzyme cleaves DNA at a specific


nucleotide sequence, hence are used experimentally to obtain defined DNA segments called restriction fragments.

Most powerful tools in molecular biology. Recombinant


DNA would not exist without the availability of these enzymes.

Nucleases
3
5 Exonuclease

5
3 Exonuclease

Endonuclease

Type II RE (93%) are ideal for Molecular


Biology

Restriction activity with modification


activity (protective for the host) present on different subunit.

Each RE cuts in a predictable and


consistent manner, at a site within the recognition sequence.

Only require Mg++ as a cofactor, ATP is not


needed.

Nomenclature of RE
One of the first type II RE characterized was from the bacterium E. Coli, and was designated EcoR1 . Names reflect origin based on genus and species

EcoR1
E = genus Escherichia co = species coli R = strain RY13 1 = first endonuclease identified

Characteristics of Type II RE
Restriction enzymes recognize and make a cut within
specific palindromic sequences, known as restriction sites, in the DNA. This is usually a 4 or 6 base pair sequence.

CIVIC,
5
3

MADAM
3 5

GAATTC GAATTC

A palindromic sequence in DNA is one in which the 5 to


3 base pair sequence is identical on both strands.

The cut site is always the same. Cleave the


phosphodiester bond (back bone of the DNA molecule) that joins adjacent nucleotides in a DNA strand.

EcoR1
EcoR1 is a restriction enzyme that searches the DNA molecule until it finds the sequence ( - GAATTC - ) of six nitrogen bases.

Cohesive Ends (Staggered cuts)

GAATTC
GAATTC
EcoR1
(Cuts between GA )

AATTC
G

AATTC

HAE III
HaeIII is a restriction enzyme that searches the DNA molecule until it finds the sequence ( -GGCC - ) of four nitrogen bases.

Blunt ends (Direct cuts) GAGGCCAG CTGGCCTC


(Cuts between GC )

GAGG

CCAG

CTGG

CCTC

-GC-GC-GG-AA-

-GA-

Frequency of cutting
Average distance between cuts is: 4n where n is number of bps in recognition site.

4-base cutter: 44 = 5-base cutter: 45 = 6-base cutter: 46 =

256 bp 1,024 bp 4,096 bp

4-base cutter: Cuts DNA into 256 bp average-sized fragments in a random sequence

Restriction Modification Activity

In addition to an endonuclease cutting activity, some of


RE also possess a modification activity that is protective for the host. Some have methylase activity that protect the host DNA from digestion by adding methyl groups to a nucleotide within the sequence recognized by the restriction enzyme. Block the recognition site.

Restriction Modification Activity

Gel Electrophoresis
Separates DNA fragments on the basis of charge and size. The larger the fragment, the more difficulty it has moving
through gels. By placing DNA in a gel, then applying a voltage across the gel, the negatively charged DNA will move toward the positive pole. Large fragments lag behind while small fragments move through the gel relatively rapidly.

Agarose gel electrophoresis - (300 bp - 15 kb)

Polyacrylamide gel electrophoresis (PAGE )- (1-500 bp)

Importance of Restriction enzymes


Analysis of chromosomes and their structure.
Sequencing very long DNA molecules.

Isolating genes. Creating new DNA molecules for cloning. DNA fingerprinting.

DNA fingerprinting
Because restriction endonucleases cut specific
sequences they can be used to make DNA fingerprints of different samples of DNA. DNA fingerprinting can help solve crimes, paternity tests, identifying bodies, etc.

DNA fingerprinting
STEPS: 1. Sample DNA cut with restriction enzymes 2. Fragments separated by size using gel electrophoresis 3.The pattern of bands produced is the DNA fingerprint, which is distinguished statistically form other individuals

Recombinant DNA

Recombinant DNA

GAATTC

AATTC

EcoR1

DNA Ligase

G AATTC
Recombinant DNA

G AATTC

AATTC

GAATTC

The Specific Cutting and Ligation of DNA


GAATTC
CTTAAG

GAATTC
CTTAAG

EcoRI
G AATTC
G

AATTC
G

CTTAA

CTTAA

AATTC

AATTC

CTTAA
EcoRI sticky end

CTTAA

G
EcoRI sticky end

DNA Ligase
G AATTC CTTAA G

Recombinant DNA

Recombinant DNA Technology


Restriction enzymes cut DNA at specific points (the
scissors)

DNA ligase pastes the DNA fragments together (the


glue)

The result is recombinant DNA Recombinant DNA, in which genes from two different
sources - often different species - are combined in vitro into the same molecule.

These methods form part of genetic engineering, the


direct manipulation of genes for practical purposes DNA technology has launched a revolution in biotechnology, the manipulation of organisms or their components to make useful products.

BACTERIA AS TOOLS FOR MANIPULATING DNA


Bacterial plasmids can serve as carriers (vectors) for gene transfer. A vector is a replicating unit that can be opened to insert another DNA fragment of interest.
Plasmids are small circular DNA molecule separate from the bacterial chromosome.

1.Isolate DNA from two sources

Bacteria take the recombinant plasmids and reproduce

This clones the plasmids and the genes they carry


Products of the gene can then be harvested

2.Cut both DNAs with the same restriction enzyme 3. Mix the DNAs; they join by basepairing with DNA ligase Recombinant DNA plasmid

The process of cloning a human gene in a bacterial plasmid can be divided into five steps.
Bacterial clone carrying many copies of the human gene.

4. Put plasmid
into bacterium

5.Clone the bacterium

POLYMERASE CHAIN REACTION (PCR)

Polymerase Chain Reaction


Developed in 1984 by Karry Mullis (Nobel Prize, 1993), PCR is now considered as a basic tool for the molecular biologist.
The polymerase chain reaction (PCR) is in vitro technique for generating large quantities of a specified DNA. PCR is a cell-free amplification technique for synthesizing multiple identical copies of any DNA of interest.

For PCR technique, it is essential to have the knowledge of the nucleotide sequence of short segments ( about 20 nucleotides), known as flanking sequences, at each end of target DNA. Steps of PCR 1. Denaturation 2. Renaturation or Annealing 3. Synthesis (chain extension)

Repeated

These three steps are repeated again and again to generate multiples of target DNA.

Materials required for PCR


The target DNA fragment. Two primers, each about 20 bases long with sequence complementary to the sequence immediately adjacent (flanking sequences) to the DNA segment of interest. DNA polymerase (Taq polymerase) which can sustain high temperature (> 60o C).

A large number of free deoxynucleotides (dNTPs) i.e dATP, dCTP, dGTP, and dTTP.

1. Denaturation: On raising the temperature to about 95 C the DNA gets denatured and the two complementary strands separate.

2
2. Renaturation or annealing : As the temperature of the mixutre is slowly cooled to about 550 - 600 C, the primers base pair with the complementary regions flanking target DNA strands. This process is called renaturation or primer annealing.

3. Synthesis (chain extension): In the presence of DNA polymerase (Taq polymerase) the primers are extended by joining the dNTPs complementary to the target DNA strand. The temperature is raised to 720C which is the optimal temperature for Taq polymerase.

3
The above process is repeated. The number of copies doubles in each cycle. 25 to 30 cycles are sufficient for effective DNA amplification.

Applications of PCR
PCR is highly sensitive, it can detect even the presence of single molecule of DNA.
1. PCR in clinical diagnosis Prenatal diagnosis of inherited diseases : PCR is employed in the prenatal diagnosis of inherited diseases by using chorionic villus samples or cells from amniocentesis. Sickle-cell anemia, thalassemia and phenylketonuria can be detected by PCR. Diagnosis of retroviral infections: PCR from is a valuable tool for diagnosis of retroviral infections, e.g., HIV infection.

Diagnosis of bacterial infections:

PCR is used for the detection of bacterial infections e.g., tuberculosis by Mycobacterium tuberculosis.

Diagnosis of cancers: Several virally-induced cancers (e.g., cervical cancer caused by human papilloma virus) can be detected by PCR.

2. Detection of criminals in forensic medicine.

3. Study of evolution from DNA of archeological samples.

DNA SEQUENCING

DNA SEQUENCING
DNA Sequencing means finding the order of nucleotides
on a single strand of DNA .
Nucleotide order determines Amino acid order, and hence protein structure and function. An alteration in a DNA sequence can lead to an altered or non functional protein leading to harmful effects. Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment.

Methods of DNA Sequencing


There are two main methods of DNA sequencing: Maxam & Gilberts Method: Chemical method of sequencing.
Sangers Method: Enzymatic or Biosynthetic method using dideoxynucleotides (ddNTPs). Modern sequencing equipment principles of the Sangers technique. uses the

Uses dideoxynucleotides i.e ddNTPs (dideoxyadenine,


dideoxyguanine, etc).

The Sangers Technique

Dideoxynucleotides resemble (analogues) normal


nucleotides but lack the normal -OH group at the 3 position as a result of which nucleotides cannot join the growing DNA chain and replication stops.

5' 3'
Normal Linking

1
OH

5'
2-

PO4
Phosphodiester bond

3'
dideoxynuceotide

P 1 R P 2 ddNTP R P R P A R P 2 R P H H R
Can not react

5'

A
Terminated

3'

Enzymatic or Biosynthetic method

Sanger's Method:
ATCG TAGCTAGCTA

32P

Keep on going

ATCG TAGCTAGCTA

Template = ddNTPs

32P

A,T,C,G

Analogue
or

ATCGA TAGCTAGCTA
Producing various fragments
STOP

Terminated

Requirements
Multiple copies of template DNA strand. A radioactive tagged primer (a small piece of DNA that
can pair with the template DNA to act as a starting point for replication).

DNA polymerase (an enzyme that copies DNA) adding


new nucleotides to the 3 end of the template

All four deoxynucleotides (dNTPs) i.e dATP, dGTP, dCTP


and dTTP

A small proportion of each of the four labeled


dideoxynucleotides (ddNTPs) . One for each of the four reactions.

Process of DNA Sequencing


Double-stranded DNA is denatured and a radioactive primer is
annealed to the DNA .

Four separate reactions are performed to synthesize new


DNA, each reaction contains all four deoxynucleotides (dNTPs) and a small portion of one of the dideoxynucleotide bases (ddNTPs).

DNA is synthesized, terminating each time a ddNTP is


incorporated.

DNA from all four reactions is separated on a gel in side-byside lanes to produce a sequence ladder. Each reaction generates a set of unique fragment lengths ending with a particular dideoxynucleotide.

The sequence is read from the bottom up, and is the


compliment (opposite) of the base identified in the gel.

DNA sequencing technology requires gel electrophoresis system with the ability to separate DNA fragments that separate by one b.p. An advancement of the sangers method is the use of automated sequencing machines using ddNTPs that are each tagged with a different color fluorescent dye.

Instead of performing four different reactions, DNA synthesis occurs in one tube. The sequencing machine then uses a light sensor to read the gel, identifying the bases by their different colors.

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