BIO 002 - BIOTECHNOLOGY

Biotechnology

The use of theories and concepts in Biological Sciences to improve our quality of life.

Medicine, Agriculture, Biofuels, Genetics

The areas where Biotechnology is mostly used are in the fields of:

BT Plants

One good example of a product of Genetic Engineering is the production of ________________

Bacillus Thuringiensis

BT stands for the bacteria ________ ____________. This is a type of bacteria that lives in the soil. These bacteria are producing proteins that are harmful to insects especially several species of Moths and Butterflies

Genetically Modified Organism

GMO Stands for:

donor

The cell or organism where the target DNA can be found is called a _______

Restriction Enzymes

Used to cut DNA into fragments

4-8

Restriction sites are naturally, _ to _ bases which are palindromic in nature

Sticky and Blunt

Restriction enzvmes may leave _______ ends or ________ ends.

DNA Cloning Vectors

Carry target gene into a host cell, in example, Plasmids and Bacteriophage

Plasmid

________ is circular double stranded DNA in a bacteria. It is different from the chromosome. Though, this carry-out DNA molecules, they are not capable of manifesting the trait nor produce the protein

Host Cell

A bacterial cell that allows the cloning vector to replicate within it. This should be non-pathogenic, harmless microorganism which is easy for cultivation

Modifying Enzymes

Introduces minor bases into DNA or RNA e.g, DNA Ligase and Taq Polymerase

Taq Polymerase

Replaces DNA Polymerase in PCR

Cloning

A molecular biology technique that makes many identical copies of a piece of DNA, such as a gene. In a typical _______ experiment, a target gene is inserted into a circular piece of DNA called a plasmid.

Molecular Cloning and Reproductive Cloning

There are two types of DNA Cloning:

extract first the DNA from the cell/DNA Extraction

Before you start with cloning, you have to ___________________________

Stage 1: Isolation of target gene

This process can be done using: (1) cutting the gene from a complete chromosome using a restriction enzyme or (2) producing a complementary DNA (cDNA). To do this the DNA must first be cut into fragments and the one containing the desired gene must be identified. Remember to use the same restriction enzyme

Stage 2: Insertion of a target gene into a vector

Gene inserted into the vectors such as plasmids and bacteriophages resulting in Recombinant DNA. Bacterial plasmids that have been isolated from bacterial cells must be mixed with the same restriction enzymes used to cut the DNA molecule. This is done to produce the same sequence of sticky ends

Stage 3: Introduction of vector into a host

The plasmids carrying the target gene must be introduced into a host cell through transformation, and only a lesser amount will contain the recombinant plasmid.

Stage 4: Amplification of the target gene by the cell (cloning) host ad screening

Following the introduction of the recombinant plasmids, bacteria will then be cultured in a medium. The transformed bacteria will be able to grow and form colonies on the medium. The bacteria will divide to produce new identical bacterial cells. Each time the bacterial cells divide, the recombinant plasmids will produce multiple copies of the target gene.

Polymerase Chain Reaction

PCR Stands for:

Polymerase Chain Reaction

A method used to amplify DNA, it resembles replication but is carried out in a test tube

Thermocycler, Test tube containing the DNA sample, Taq polymerase, primers, free DNA nucleotides

Tools used in PCR

Thermal Cycler or thermocycler

A laboratory instrument that heats and cools samples in repetitive cycles to facilitate DNA or RNA amplification through the polymerase chain reaction.

Step 1: Denaturation

Double-stranded DNA templates are heated to separate the strands; The reaction temperature is increased to 95 °C, which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into singlestranded DNA (ssDNA)

Step 2: Annealing

The DNA molecule will be exposed at a lower temperature to allow the primers to attach to the Single-Strand DNA.

Primer

The role of the _______ is to let Taq Polymerase copy the segment.

Taq Polymerase

____ __________ is an enzyme derived from a bacteria Thermus aquaticus which mimics the job of DNA Polymerase but can withstand a high temperature

Step 3: Extension

The temperature will be raised again Taq Polymerase will copy and texted the segment of DNA.

1 Cycle, 4 Copies of DNA

after the third step, it is considered that 1 cycle is complete and there is 22 amount of DNA in the container (usually an Eppendorf). So after __ cycle we can assume that there is __ copies of DNA.

Taq polymerase is heat-stable form that can function after exposure to the high temperatures that are necessary for PCR.

Why is taq polymerase used in PCR instead of DNA polymerase?

CRISPR

The most common gene editing tool is

clustered regularly interspaced short palindromic repeats

CRISPR Stands for

Bacteriophages

Restriction enzvmes are based from bacteria that are immune to _______________

Plasmid or Bacteriophage

What is the vector used in genetic engineering

because it will produce fragments with the same complementary sticky ends

Why is it important to use the same restriction enzyme?

Cytosine, Guanine, Adenine, Thymine

What is the language of DNA?

Primer

The role of the _______ is to let Taq Polymerase copy the segment