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CHAPTER REVIEW
Recombinant DNA Technology Recombinant dna tecnology
rDNA technology
Tools
RE & Palindromic Steps in gene sequence Types Cloning vector & characteristics
cloning
PCR
Presents in a chromosome cell and contains genetic information for all living organisms.
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.
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.
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.
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
small rings / circular of double stranded DNA molecules that carry genes separate from the main bacterial chromosome
Plasmids
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
Can replicate independently. - So any gene added to a plasmid is also copied many times ( cloned ) once it is inside a host cell
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)
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
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.