Paper X

Dr. Sajjan M. B.

Asst. Prof. in Zoology,

Raje Ramrao Mahavidyalaya, Jath

�Unit 3 : Molecular Techniques in Gene manipulation 15�

• 1. Restriction Enzymes

2. Characteristics of Cloning vectors: Plasmids, Cosmids, Phagemids, Lambda Bacteriophages�

• 1. Introduction
• 2.Need
• 3.Characters
• 4. Types
• Use of - Plasmids, Cosmids, Phagemids, Lambda Bacteriophages�

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3. Gene cloning: Transformation techniques by Calcium chloride method and electroporation

• 1.Introduction
• 2. Transformation technique
• 3. Calcium chloride method
• i. Procedure
• ii. Applications
• iii. Limitations

2. Artificial competence

• . Bacteria can also be made competent artificially by
• chemical treatment and
• heat shock

to make them permeable to DNA.

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Competence and Transformation�

• Artificial competence : This procedure is comparatively easy and simple, and can be used in the genetic engineering of bacteria but in general transformation efficiency is low. 
• Methods for preparing the competent cells derive from the work of Mandel and Higa who developed a simple treatment based on soaking the cells in cold CaCl2. There are two main methods for the preparation of competent cells.
• They are: 1.Calcium chloride method
• 2. Electroporation.

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1.Calcium chloride method �

• Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which foreign DNA can be passed through easily.
• Most types of cells cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatments to make them competent.
• The standard method for making the bacteria permeable to DNA involves treatment with calcium ions.

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Transformation procedure by CaCl2�

1. The CaCl2 treated competent cells are taken .

2. Plasmid containing DNA was directly pipetted over competent cells.

3. These cells were mixed gently by tapping 4-5 times,

4. Incubated on ice for 30 min.

5. which was followed by a heat shock treatment.

Procedure

• ln CaCl2 method, the competency can be obtained by creating pores in bacterial cells by 1.suspending them in a solution containing a high concentration of calcium.
• DNA can then be forced into the Host cell by 2. heat shock treatment at 42oC for the process of transformation

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• As DNA is a highly hydrophilic molecule, normally it cannot pass through the cell membrane of bacteria.
• Divalent cations generate coordination complexes with the negatively charged DNA molecules and LPS (Lipopolysaccharides).
• DNA, being a larger molecule, cannot itself cross the cell membrane to enter into the cytosol.

• The heat shock step strongly depolarizes the cell membrane of CaCl2-treated cells, so that membrane potential decreases.
• Thus, the decrease in membrane potential lowers the negativity of the cell’s inside, which ultimately allows the movement of negatively charged DNA into the cell’s interior.
• 3. The subsequent cold shock again raises the membrane potential to its original value.

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• Rapidly growing cells are made competent more easily than cells in other Growth stages.
• So it is necessary to bring cells into log phase before the procedure is begun.
• The cells in rapid growth (log phase) are living, healthy, and actively metabolizing.
• Competent cells are readily available in commercial markets.

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Electroporation

• Electroporation is a technique involving electric field mediated membrane permeabilization.
• Electric shock is given to cells, pores are formed in the cell membrane.Electric shocks can also induce cellular uptake of exogenous DNA through these pores.
• Electroporation is a simple and rapid technique for introducing genes into the cells from various organisms(microorganisms, plants and animals)

Electroporation technique

• The basic technique of electroporation for transferring genes in to mammalian cells :
• 1. The cells are placed in a solution containing DNA .
• 2. They are subjected to electric shocks to cause holes in the membranes.
• 3. The foreign DNA fragments enter through the holes in to the cytoplasm and then to nucleus.

Limitation �

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• Under normal conditions, the amount of DNA delivered into plant cells is very low.
• Efficiency of electroporation is highly variable depending on the plant material and the treatment conditions.

Construction of genomic and cDNA libraries �

• 1. Introduction
• 2. Genomic library
• 3. c-DNA library
• 3. Steps
• 4. Applications

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Application

• Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs,
• developmental stages,
• environmental stress levels,
• pathogen infection, and over the course of treatment.

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6. DNA sequencing: Sanger method�

6. DNA sequencing: Sanger method�

• 1. Introduction
• 2. Principle
• 3. Requirements
• 4. Method

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7. Polymerase Chain Reaction,

• 1.Introduction
• 2. Principal
• 3. Technique
• i) Denaturation
• ii) Renaturation
• iii) Synthesis
• 4. Significance
• 5. Medical applications

Amplification of DNA – Why, How?

• Some microorganisms, viruses have small DNA molecule, it is difficult to study it and analyse it.
• How to amplify genes in to millions of copies producing enough DNA copies to be analyzed 
• DNA associated with genetic disorders from the DNA of patients (or from fetal DNA, in the case of prenatal testing).

How to carry out procedure in vitro ?

• Step1 : in vitro Denaturation of double stranded DNA requires high temperature, about 95degree C.
• Step 2 : Pairing of primers with template requires low temp., about 55degree C.
• Step 3: DNA polymerase enzyme should withstand high temp., about 95 degree C.

Discovery - Karry Mullis

• Scientists discovered DNA polymerase enzyme from the bacteria Thermus aquaticus found in hot spring (90-100degree).
• This thermostable enzyme is known as Taq DNA polymerase.
• It can work at high temp.
• For repeated cycles of DNA replication a machine is invented

Repeated cycles

• 1. 20 – 40 repeated cycles of DNA replication.
• 2. After each cycle molecules of DNA are doubled.
• 3. From 40 cycles 1 trillion copies are produced from a single copy of DNA molecule.

• 1. Fast and inexpensive method of amplification of DNA
• 2.It is a Molecular photocopying

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DNA finger printing

DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics.

DNA profiling is a forensic technique in criminal investigations, comparing criminal suspects' profiles to DNA found as the evidence so as to assess the possibility of their involvement in the crime.

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 It is also used in parentage testing, to establish immigration eligibility, and in genealogical and medical research. DNA profiling has also been used in the study of animal and plant populations in the fields of zoology, botany, and agriculture.

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• A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.
•  Each DNA spot contains picomoles (10⁻¹² moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element 

• It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. Also for identification of structural variations and measurement of gene expression

Oligonucleotide based arrays

• Oligonucleotides are sequences of few nucleotides.
• They are designed to match part of target DNA.