CHAPTER 22

22.1 RECOMBINANT DNA TECHNOLOGY

CHAPTER REVIEW
Recombinant DNA Technology Recombinant dna tecnology

Application of Recombinant DNA Technology

rDNA technology
Tools

Methods in gene cloning

Agriculture Production of insulin Environment

RE & Palindromic Steps in gene sequence Types Cloning vector & characteristics
cloning

PCR

Host cell (bacteria) & characteristics Modifying enzyme & functions

By the end of this topic, should be able to:

Define recombinant DNA technology

State the tools used in recombinant DNA technology

Explain restriction enzyme and examples

List and explain the types of cloning vectors

Presents in a chromosome cell and contains genetic information for all living organisms.

RECOMBINANT DNA TECHNOLOGY

Special techniques produced from genetic engineering process. Technology that produced any DNA molecule made in vitro with segments from different sources / by combining genes from different organisms.

RECOMBINANT DNA TECHNOLOGY

A new field of biology - started in the mid 1970s Permits the formation of new combinations of genes By isolating genes from the organisms And introducing them into either a similar or unrelated organism.

RECOMBINANT DNA TECHNOLOGY

By this method: a.foreign DNA may be inserted not only in the simple single cells of bacteria but also into cells from the bodies of complex organisms b.once inside the cells, this foreign DNA may be expressed.

TOOLS USED IN DNA RECOMBINANT TECHNOLOGY

Target DNA (gene of interest) DNA ligase (modifying enzyme)

DNA cloning vector

Restriction enzyme

Host cell

RESTRICTION ENZYME

RESTRICTION ENZYME

Class of enzyme that recognizes specific base sequences of DNA and cut / cleaves the DNA molecular at that side. Today, more than 800 different types known

RESTRICTION ENZYME

RESTRICTION ENZYME

In natural, these enzymes protect the bacteria against intruding (interfering) DNA/RNA from another organism like viruses. The bacterial DNA was modified, so that it was immuned to its own restriction enzyme

RESTRICTION ENZYME
Characteristics

Have ability to splice through DNA at specific sequence different restriction enzyme cut a particular base sequences, called restriction sites.

RESTRICTION ENZYME
Characteristics

PALINDROME

The base sequences of one strand reads the same as its compliment strands

RESTRICTION ENZYME
Characteristics

palindrome
SPELL THIS!

M A D A M

M A D A M

RESTRICTION ENZYME
Characteristics

Example :
5 GAAT T C C T TAAG 3 5 3

RESTRICTION ENZYME
Characteristics

RE hydrolyzes the phosphodiester backbone of each strand RE can produce two types of cutting pattern : sticky end (EcoRI) blunt end (SmaI)

RESTRICTION ENZYME
Characteristics

EcoRI

cutting pattern

RESTRICTION ENZYME
Characteristics

SmaI

cutting pattern

Several types of RE

CLONING VECTOR

CLONING VECTOR carrier DNA into which a DNA fragment containing a particular gene can be inserted, and which is then used to introduce gene into a new organism or DNA that carries gene of target into the host cell

DNA cloning vectors

Plasmids

small rings / circular of double stranded DNA molecules that carry genes separate from the main bacterial chromosome

small (smaller than 10 kb base pairs)

DNA cloning vectors

Plasmids

Plasmids

DNA cloning vectors

usually carry only one of a few genes and have a single origin of replication. genes on plasmid often encode protein (eg : enzyme) that protect the bacterium from one or more antibiotics easily isolated from a bacterium can be reintroduced to another bacterial cell able to express cloned genes able to clone up about 8 kb pieces of DNA

Plasmids

DNA cloning vectors

Plasmids is extracted by breaking up the cells

and separating the contents by centrifugation.

Once isolated, the plasmid rings are cut open

enzymically, using restriction enzyme.

Can replicate independently. - So any gene added to a plasmid is also copied many times ( cloned ) once it is inside a host cell

Plasmid combine with DNA target forming recombinant DNA.

DNA cloning vectors

Bacteriophage Phage is a virus that infects bacteria Constructed from phage lambda chromosome e.g. 2001

Size of DNA fragment: can handle up from 37 to 52 kb

Phage DNA can accept foreign DNA restriction fragment (forming rDNA) Phage infects E.coli (host cell) by injecting its rDNA rDNA replicates in E.coli (producing rDNA copies)

DNA cloning vectors

Bacteriophage

Cosmid
Hybrids between plasmids and phage lambda chromosome. Packaged into capsule of phage Can accept larger DNA restriction fragments Suitable for cloning large mammalian genes or multigene fragments Normal cosmid are able to carry only 10-15 K base pair E.g. sCOS-1

DNA cloning vectors

Yeast artificial chromosome (YAC)

DNA cloning vectors

a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). these chromosomes behave normally in mitosis and can carry more DNA than a plasmid. artificially constructed chromosome - contains telomeric (specialized nucleoprotein complexes that have important functionsreplication), centromeric, and replication origin sequences needed for replication and preservation in yeast cells.