1. Genetic Engineering/ Recombinant DNA Technology
General Genetics
Dr. Attya Bhatti

2. Genetic Engineering Also known as Gene manipulation
Genetic modifications
recombinant DNA technology,
New Genetics
Means:
altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.
Various kinds of genetic modification are possible:
inserting a foreign gene from one species into another,
forming a transgenic organism
altering an existing gene so that its product is changed
Changing gene expression so that it is translated more often or not at all.

3. History of Genetics Since 1900.
Mendelian Genetics
1990
1910
1920
1930
1940
1950
1960
1970
1980
2000
Genetic Mapping
Transformation demonstration
Microbial Genetics
Molecular Genetics
Gene Manipulation
Development of techniques
Applications

4. Basic Concepts of Genetic Engineering
Recombinant DNA technology is a set of methods used to locate, analyze, alter, study, and recombine DNA sequences.
It is used to probe the structure and function of genes, address questions in many areas of biology, create commercial products, and diagnose and treat diseases.

5. Steps in Genetic Engineering
Isolate the gene
Insert it in a host using a vector
Produce as many copies of the host as possible
Separate and purify the product of the gene
Generation of DNA Fragments
Joining to a vector or carrier Molecule
Introduction into a host cell for amplification
Selection of required sequence.

6. Step 1: Isolating the Gene

7. Step 1: Alternative Method (using reverse transcriptase)
mRNA converted into cDNA
Complementary strand produced using DNA polymerase
Advantage – more mRNA in cell than DNA

8. Step 2: Inserting Gene into Vector
Vector – molecule of DNA which is used to carry a foreign gene into a host cell

9. Step 3: Inserting Vector into Host

10. Replica Plating

11. Step 4: Multiplication of the Host Cells by Cloning
Large scale fermenters by cloning
All genetically identical because of asexual reproduction

12. Step 5: Extraction of desired gene product.

13. Genetically engineered corn, which produces a toxin that kills insect pests, now comprises over 30% of all corn grown in the United States.
Recombinant DNA technology has been used to create genetically modified crops.

14. Working at the Molecular Level
Recombinant DNA technology requires special methods because:
Individual genes make up a tiny fraction of the cellular DNA and they cannot be seen.

15. Recombinant DNA Techniques
Methods for locating specific DNA sequences:
Techniques for cutting DNA at precise locations
Procedures for amplifying a particular DNA sequence billions of times, producing enough copies of a DNA sequence to carry out further manipulations
Methods for mutating and joining DNA fragments to produce desired sequences
Procedures for transferring DNA sequences into recipient cells

16. Restriction Enzymes
Also called restriction endonucleases that recognize and make double-stranded cuts in the sugar–phosphate backbone of DNA molecules at specific nucleotide sequences.
These enzymes are produced naturally by bacteria, where they are used in defense against viruses.
In bacteria, restriction enzymes recognize particular sequences in viral DNA and then cut it up. A bacterium protects its own DNA from a restriction enzyme by modifying the recognition sequence, usually by adding methyl groups to its DNA.

17. Types of Restriction Enzymes
Three types of restriction enzymes have been isolated from bacteria
Type I restriction enzymes
Type II restriction enzymes
Type III restriction enzymes

18. Type I Restriction Enzymes
Recognize specific sequences in the DNA
Cut the DNA at random sites that may be some distance (1000 bp or more) from the recognition sequence

19. Type II Restriction Enzymes
Recognize specific sequences
Cut the DNA within the recognition sequence
Virtually all work on recombinant DNA is done with type II restriction enzymes

21. Type III Restriction Enzymes
Recognize specific sequences
Cut the DNA at nearby sites
Usually about 25 bp away

23. The number of restriction sites is related to the number of fragments produced when DNA is cut by a restriction enzyme

24. Vectors
Is a vehicle for delivering genetic material such as DNA to a cell
Cloning vectors
Plasmid vectors
Bacteriophage vectors

25. Cloning Vectors
is a stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell.
Three important characteristics:
an origin of replication which ensures that the vector is replicated within the cell.
Selectable markers, which enable any cells containing the vector to be selected or identified.
one or more unique restriction sites into which a DNA fragment can be inserted.

26. Three characteristics of an idealized cloning vector

27. Plasmid vectors
Plasmids are circular DNA molecules that exist naturally in bacteria
contain origins of replication and are therefore able to replicate independently of the bacterial chromosome
Used in cloning have been constructed from the larger, naturally occurring bacterial plasmids

28. Plasmid vectors Example: pUC19 plasmid has an origin of replication
two selectable markers—an ampicillin-resistance gene and a typical cloning vector

29. The pUC19 plasmid is a typical cloning vector

30. Bacteriophage Vectors
Bacteriophages offer a number of advantages as cloning vectors.
Most widely used bacteriophage vector is bacteriophage , which infects E. coli
Advantages:
High efficiency with which it transfers DNA into bacteria cells

31. Viewing DNA Fragments
DNA fragments can be separated, and their sizes can be determined with the use of gel electrophoresis.
The fragments can be viewed by
Using a dye that is specific for nucleic acids
By labeling the fragments with a radioactive or chemical tag.

32. Gel electrophoresis can be used to
separate DNA molecules on the basis of their size and electrical charge

33. Cloning Genes
Identical copies (clones) of the original piece of DNA are produced
DNA fragments can be inserted into cloning vectors, stable pieces of DNA that will replicate within a cell.
Cloning vectors must have an origin of replication, one or more unique restriction sites, and selectable markers.
Plasmids are commonly used as cloning vectors.

35. Applications Basic Research on gene Structure and Function
Production of useful proteins by novel methods
Generation of transgenic plants and animals
Medical diagnostics and treatment

36. Applications
In addition to providing valuable new information about the nature and function of genes, recombinant DNA technology has many practical applications
Include the production of pharmaceuticals and other chemicals, specialized bacteria, agriculturally important plants, and genetically engineered farm animals

37. Applications Oligonucleotide Drugs:
Oligonucleotide drugs are short pieces of DNA or RNA that prevent the expression of particular genes.
Genetic Testing:
The identification and cloning of many important disease causing human genes has allowed the development of probes for detecting disease-causing mutations.

38. Applications Genetic Testing:
The identification and cloning of many important disease causing human genes has allowed the development of probes for detecting disease-causing mutations.
Gene Therapy:
Ultimate application of recombinant DNA technology
is gene therapy the direct transfer of genes into humans to treat disease

39. Applications Gene Mapping:
Significant contribution of recombinant DNA technology has been to provide numerous genetic markers that can be used in gene mapping.
One group of markers used in gene mapping comprises restriction fragment length polymorphisms (RFLPs, pronounced rifflips).

40. Applications DNA Fingerprinting:
Restriction fragment length polymorphisms are often found in non coding regions of DNA and are therefore frequently quite variable in humans.