Techniques in Molecular Biology (COMPLETE)

7/29/2019 Techniques in Molecular Biology (COMPLETE)

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MOLECULAR BIOLOGY TECHNIQUES

Dr. Mohammed Shakil Akhtar


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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.


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Restriction endonucleases (restriction enzymes) cleave

double-stranded DNA into smaller, more manageablefragments.

Each restriction enzyme cleaves DNA at a specificnucleotide sequence, hence are used experimentally toobtain defined DNA segments called restriction fragments.

Most powerful tools in molecular biology. Recombinant

DNA would not exist without the availability of these

enzymes.

Restriction Endonucleases (RE)


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Nucleases

Endonuclease

5 Exonuclease 3 Exonuclease

5 533


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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.


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One of the first type II RE characterized was from the

bacterium E. Coli, and was designated EcoR1 .

Nomenclature of RE

Names reflect origin based on genus and species

EcoR1

E = genus Escherichia

co= species coliR= strain RY13

1= first endonuclease identified


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Characteristics of Type II RE

GAATTC

GAATTC

5

5

3

3

A palindromic sequence in DNA is one in which the 5 to3 base pair sequence is identical on both strands.

Restriction enzymes recognize and make a cut withinspecific palindromic sequences, known as restriction

sites, in the DNA. This is usually a 4 or 6 base pair

sequence.

CIVIC, MADAM


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The cut site is always the same. Cleave thephosphodiester bond (back bone of the DNA molecule) that

joins adjacent nucleotides in a DNA strand.


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GAATTC

GAATTC

G

AATTC G

AATTC

Cohesive Ends

(Staggered cuts)

EcoR1

EcoR1

EcoR1 is a restriction enzyme that searches the DNAmolecule until it finds the sequence ( - GAATTC - ) of

six nitrogen bases.

(Cuts between GA)


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HAE III

HaeIII is a restriction enzyme that searches the DNA

molecule until it finds the sequence ( -GGCC - ) of fournitrogen bases.

Blunt ends

(Direct cuts)

GAGGCCAG

CTGGCCTC

GAGG

CCTC CTGG

CCAG

(Cuts between GC)


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-GC-

-GC-

-GG-

-AA-

-GA-


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Average distance between cuts is: 4n

where n is number of bps in recognition site. 4-base cutter: 44 = 256 bp

5-base cutter: 45 = 1,024 bp

6-base cutter: 46 = 4,096 bp

Frequency of cutting

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


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In addition to an endonuclease cutting activity, some ofRE also possess a modification activity that is protective

for the host.

Restriction Modification Activity

Some have methylase activity that protect the host DNAfrom digestion by adding methyl groups to a nucleotide

within the sequence recognized by the restriction enzyme.

Block the recognition site.


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Restriction Modification Activity


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Gel Electrophoresis

The larger the fragment, the more difficulty it has movingthrough 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 throughthe gel relatively rapidly.

Separates DNA fragments on the basis of charge and size.

Agarose gel electrophoresis - (300 bp - 15 kb)

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


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Analysis of chromosomes and their structure.

Importance of Restriction enzymes

Sequencing very long DNA molecules.

Isolating genes.

Creating new DNA molecules for cloning.

DNA fingerprinting.


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Because restriction endonucleases cut specificsequences they can be used to make DNA fingerprints

of different samples of DNA. DNA fingerprinting can helpsolve crimes, paternity tests, identifying bodies, etc.

DNA fingerprinting


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STEPS:

1. Sample DNA cut with restriction enzymes

2. Fragments separated by size using gelelectrophoresis

3.The pattern of bands produced is the DNA fingerprint,

which is distinguished statistically form other

individuals

DNA fingerprinting


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Recombinant DNA


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GAATTC

GAATTC

G

AATTC G

AATTC

EcoR1

GAATTC G

AATTC

DNA Ligase

Recombinant DNA

Recombinant DNA

Th S ifi C i d Li i f DNA


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The Specific Cutting and Ligation of DNA

GAATTC

CTTAAG

GAATTC

CTTAAG

G

CTTAA

AATTC

G

AATTC

G

G

CTTAA

G

CTTAA

AATTC

G

G

CTTAA

AATTC

G

G

CTTAA

AATTC

G

EcoRI

DNA LigaseEcoRI sticky end EcoRI sticky end

Recombinant DNA


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Recombinant DNA Technology

Recombinant DNA, in which genes from two differentsources - often different species - are combined in vitro

into the same molecule.

Restriction enzymes cut DNA at specific points (thescissors)

DNA ligase pastes the DNA fragments together (theglue)

The result is recombinant DNA

These methods form part of genetic engineering, thedirect 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.


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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 anotherDNA fragment of interest. Plasmids are small circular DNA molecule separate

from the bacterial chromosome.


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Bacteria take the recombinantplasmids and reproduce

This clones the plasmidsand the genes they carry

Products of the gene canthen be harvested

The process of cloning ahuman gene in a bacterialplasmid can be divided intofive steps.

1.Isolate DNAfrom two sources

2.Cut both

DNAs with the samerestriction enzyme

3. Mix the DNAs;they join by base-pairing with DNAligase

RecombinantDNA

plasmid

4. Put plasmidinto bacterium

5.Clone thebacterium

Bacterial clone carrying manycopies of the human gene.


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POLYMERASE CHAIN REACTION (PCR)


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The polymerase chain reaction (PCR) is in vitro technique for generating large quantities of a specified

DNA.

Polymerase Chain Reaction

PCR is a cell-free amplification technique for

synthesizing multiple identical copies of any DNA of

interest.

Developed in 1984 by Karry Mullis (Nobel Prize, 1993),

PCR is now considered as a basic tool for the molecular

biologist.


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For PCR technique, it is essential to have the

knowledge of the nucleotide sequence of short

segments ( about 20 nucleotides), known as flankingsequences, at each end of target DNA.

These three steps are repeated again and again to

generate multiples of target DNA.

Steps of PCR

1. Denaturation

2. Renaturation or Annealing

3. Synthesis (chain extension)

Repeated


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The target DNA fragment.

Materials required for PCR

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.


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1. Denaturation: On raising the

temperature to about 95 C the

DNA gets denatured and the twocomplementary strands separate.

1

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 iscalled renaturation or primer

annealing.

2


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3. Synthesis (chain extension): In

the presence ofDNA polymerase

(Taq polymerase) the primers are

extended by joining the dNTPscomplementary to the target

DNA strand. The temperature is

raised to 720C which is the

optimal temperature for Taqpolymerase.

3

The above process is

repeated. The number of copiesdoubles in each cycle. 25 to 30

cycles are sufficient for effective

DNA amplification.

Applications of PCR


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PCR is highly sensitive, it can detect even the presence of

single molecule of DNA.

Applications of PCR

1. PCR in clinical diagnosis

Prenatal diagnosis of inherited diseases :

PCR is employed in the prenatal diagnosis of inheriteddiseases 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.


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Diagnosis of cancers:

Several virally-induced cancers (e.g., cervical cancer caused by

human papilloma virus) can be detected by PCR.

Diagnosis of bacterial infections:

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

tuberculosis by Mycobacterium tuberculosis.

2. Detection of criminals in forensic medicine.

3. Study of evolution from DNA of archeological samples.


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DNA SEQUENCING

DNA SEQUENCING


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DNA Sequencing means finding the order of nucleotides

on a single strand of DNA .

DNA SEQUENCING

Nucleotide order determines Amino acid order, andhence protein structure and function.

An alteration in a DNA sequence can lead to an alteredor non functional protein leading to harmful effects.

Understanding a particular DNA sequence can shedlight on a genetic condition and offer hope for the

eventual development of treatment.


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There are two main methods of DNA sequencing:

Maxam & Gilberts Method: Chemical method ofsequencing.

Methods of DNA Sequencing

Sangers Method: Enzymatic or Biosyntheticmethod using dideoxynucleotides (ddNTPs).

Modern sequencing equipment uses theprinciples of the Sangers technique.

The Sangers Technique


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The Sangers Technique Uses dideoxynucleotides i.e ddNTPs (dideoxyadenine,

dideoxyguanine, etc).

Dideoxynucleotides resemble (analogues) normalnucleotides 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.


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5'


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Phosphodiester

bond

P

R

PR

P

R

P

R

P

RP

R

OH

5'

3'1

3'

5'

A

1

2

3

4

5

6

APO42-

H3'

5'

2H

dideoxynuceotide

TerminatedddNTPNormalL

inking

Can not react


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Sanger's Method:

ATCGTAGCTAGCTA

TAGCTAGCTA

TAGCTAGCTA

ATCGA

32P

ATCG

STOP

ATerminated

Keep on going

Enzymatic or Biosynthetic method

Template

or

32P A,T,C,G AAnalogue

Producing various fragments

A= ddNTPs

Requirements


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Multiple copies oftemplate DNA strand.

A radioactive tagged primer(a small piece of DNA thatcan pair with the template DNA to act as a starting pointfor replication).

DNA polymerase (an enzyme that copies DNA) addingnew nucleotides to the 3 end of the template

All fourdeoxynucleotides (dNTPs) i.e dATP, dGTP, dCTPand dTTP

A small proportion of each of the four labeleddideoxynucleotides (ddNTPs) . One for each of the four

reactions.

Requirements


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Process of DNA Sequencing


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Process of DNA Sequencing

Double-stranded DNA is denatured and a radioactive primer isannealed 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 isincorporated.

DNA from all four reactions is separated on a gel in side-by-side 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 thecompliment (opposite) of the base identified in the gel.


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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.

DNA sequencing technology requires gel

electrophoresis system with the ability to separate DNAfragments that separate by one b.p.

Instead of performing four different reactions, DNA

synthesis occurs in one tube. The sequencing machinethen uses a light sensor to read the gel, identifying the

bases by theirdifferent colors.


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Techniques in Molecular Biology (COMPLETE)

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