CHAPTER 4

DNA CLONING(cont.)

MISS NUR SHALENA SOFIAN

Steps in Gene Cloning : The use of pBR322 as vector

• Contains genes that confer resistance to antibiotics: ampicillin and tetracycline

• Also contains one cutting site for RE e.g. EcoRI, PstI, HindIII, SalI

• E.g - Cut the foreign DNA and plasmid with PstI yielding both DNA with sticky ends- combining both DNA and ligate them together

• Transform the recombinant plasmid into host E. coli

• To make the E.coli permeable to the extracellular plasmid, incubate the cells with calcium salt solution / electroporation – cells are competent

• PROBLEMS : HOW CAN ONCE SORTED OUT THE RIGHT RECOMBINANTS?– Religated plasmid– Religated insert– Recombinants

• Foreign DNA which has been cut with PstI is inserted within AmpR gene

•After transformation, cells are grown in media of tetracycline – cells with plasmid will grow

• Grow copies of the original colonies with ampicillin which kills cells with plasmid including foreign DNA

Screening With Replica Plating• Replica plating transfers clone

copies from original tetracycline plate to a plate containing ampicillin

• A sterile velvet transfer tool can be used to transfer copies of the original colonies

• Desired colonies are those that do NOT grow on the new ampicillin plate

pUC plasmids are based on pBR322 – having MCS The procedure shown seeks to clone the human β-globin

gene into a plasmid vector The vector carries two important genes

ampR Confers antibiotic resistance to the host cell lacZ Encodes β-galactosidase

Provides a means by which bacteria that have picked up the cloned gene can be identified

Steps in Gene Cloning: The use of pUC as vector

Nonrecombinant: recircularized Recombinant: vector plus inserted cloned gene Selection for vector: ampicillin resistance Selection for recombinant vs. nonrecombinant

vector: β-galactosidase activity Selection for gene of interest?

The growth media contains two relevant compounds: IPTG (isopropyl-b-D-thiogalactopyranoside)

A lactose analogue that can induce the lacZ gene X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside)

A colorless compound that is cleaved by β-galactosidase into a blue dye

The color of bacterial colonies will therefore depend on whether or not the β-galactosidase is functional

If it is, the colonies will be blue If not, the colonies will be white

In this experiment Bacterial colonies with recircularized vectors form blue colonies While those with hybrid vectors form white colonies

Note: In this case, the β-globin gene was inserted into the plasmidIt is also possible for any other DNA fragment to be inserted into the plasmidAnd it is possible for the plasmid to circularize without an insertThis is called a recircularized vector

This is termed a hybrid vector

Cells that are able to take up DNA are called competent cells

This step of the procedure is termed transformation.

PROBLEMS: cloning into pUC can give false-positive results having white colonies without inserts – vector religating itself

• Treating with alkaline phosphatase – removes the 5’-phosphates necessary for ligation

• Vector cannot religate to itself but can ligate to the inserts which retain its 5’-phosphates

The net result of gene cloning is to produce an enormous amount of copies of a gene During transformation, a single bacterial cell usually takes up a

single copy of the hybrid vector Amplification of the gene occurs in two ways:

1. The vector gets replicated by the host cell many times2. The bacterial cell divides approximately every 30 minutes

Recombinant DNA technology is not only used to clone genes Sequences such as telomeres, centromeres and highly

repetitive sequences can be cloned as well

Steps in Gene Cloning: The use of phage as vector

• A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish

• It is caused by the lysis of bacterial cells as a result of the growth and reproduction of phages

Plaques

Other phage vectors used in gene cloning

• λ phage – can accommodate large foreign DNA – usually useful in constructing genomic libraries e.g. Charon 4 λ phage can accept 20kb DNA

• Cosmids – behaving as both plasmids and phages. Having ‘cos’ sites and plasmid origin of replication. Large inserts of 40-50kb

• M13 phage – filamentous phage. DNA inserts can be in single stranded form

• Phagemids – having the same characteristics as cosmids and produce ssDNA e.g pBluescript

Eukaryotic Vectors

• Ti plasmid of Agrobacterium tumefacians carrying genes into plant cells

• YAC• BAC – designed for cloning huge pieces of DNA