Chapter 14. Genetic Engineering Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce.

Chapter 14. Genetic Engineering

Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.

Genetic modifications include:

(a) inserting a foreign gene from one species into another;

(b) forming a transgenic organism;

(c) altering an existing gene so that its product is changed;

(d) changing gene expression so that it is translated more often or not at all.

1. The most common genetic engineering techniques: cDNA: to make a DNA copy of mRNA; Restriction enzymes: to cut DNA at specific points,

making small fragments; Vectors: to carry DNA into cells and ensure replic

ation; DNA cloning: to deliver a gene to a living cell; Genetic markers: to identify cells that have been tr

ansformed; PCR: to amplify very small samples of DNA; DNA probes: to identify and label a piece of DNA

containing a certain sequence.

1) Types of recombination: recombination is a process to produce a DNA molecule by combining different segments of DNA.

(A)Homologous recombination: the recombinant DNA molecules are formed by breakage and joining of homologous segments of parent DNA duplexes.

Homologous recombination plays a key role in the repair of DNA.

Homologous recombination (Holliday Model)

(B) Site-specific recombination: is the exchange of two specific but not necessarily homologous DNA sequences.

Site-specific recombination is a mechanism to create an enormous number of different antibody molecules and T-cell receptors, e.g. the association of different V,D,J genes with C gene.

V-segment genes(~250)

D-segment genes(~15)

J-segment genes(4)

C

Electron micrograph of recombination

2) Restriction enzymes: these enzymes recognize specific nucleotide sequences in double stranded DNA and cut the double strands at specific locations.

Three types of cleavage by restriction enzymes: 5’ overhang, 3’ overhang, and blunt end.

The ends created by restriction enzymes can be ligated to form new DNA molecules, called recombinant DNA.

5’ overhang: 3’ overhang: Blunt end:

BamHI KpnI PvuII

GGATCC GGTACC CAGCTG

CCTAGG CCATGG GTCGAC

G3’ 5’GATCC GGTAC3’ 5’C CAG3’ 5’CTG

CCTAG5’ 3’G C5’ 3’CATGG GTC5’ 3’GAC

Formation of recombinant DNA

GAATTCCTTAAG

GAATTCCTTAAG

GCTTAA

AATTCG

GAATTCTTAA

AATTCG

AATTCGCTTAA

GAATTCGCTTAA

G

EcoRI

PstI

BamHI

SalI

PvuII

Using DNA ligase a foreign DNA fragment can be integrated into a plasmid DNA molecule and expressed in bacteria.

3) Electrophoresis: polyacrylamide gel electrophoresis (PAGE) is often used to separate small DNA fragments (<500bp) and agarose gel’s to separate larger DNA fragments. The bands can be visualized by staining with ethidium bromide (EB) or by labeling with radio isotope such as 32P.

Markers of standard DNA fragments of known MW are used to measure the size of the separated restriction fragments.

Gel electrophoresis

Bands of restriction fragments

Marker

4) Restriction map: refers to the sequence of restriction enzyme sites labeled on a double-stranded DNA. It may be used to compare the similarity between two DNA molecules.

E B X E S C X B

0 5 10 15 20

E: EcoRI, B: BamHI, X:XhoI, S:SalI, C:CpoI

5) Restriction fragment length polymorphism (RFLP): refers to the restriction fragment size changes that are very common in individuals in a population. These changes are produced by mutations.

RFLP analysis can be used to detect or screen for some genetic diseases such as sickle-cell anemia.

RFLP analysis for the HbS gene

Normal Sickle

14.0kb

7.7kb

H paI H paIH paI

H paI H paI

7.6kb 6.4kb

D N A probe

The advantages of RFLP analysis:

A) The results can be used to clone interested genes (e.g. those cause diseases) for sequencing or study;

B) The RFLP acts as a screening marker for some genetic diseases even in the absence of the gene, e.g. HbS gene screening.

2. Nucleic acid hybridization: refers to the formation of double-stranded structures from two single-stranded nucleic acid molecules, such as DNA-DNA, DNA-RNA, RNA-RNA.

Melting temperature (Tm): the temperature at which half of the DNA molecules have denatured. Different DNA molecules have different Tm values, depending on the content of G and C.

Renaturation (annealing): refers to the process that when the temperature falls below the Tm, the two complementary DNA strands re-form a double stranded molecule.

Denaturation

Annealing

1) DNA probe: a single-stranded DNA fragment that is complementary to the nucleic acid being assayed. The probe must be labeled such as radioisotope.

DNA probe

2) Southern blotting: consists of the following steps:

A) Agarose gel electrophoresis of restriction DNA fragments

B) Denaturation of the DNA fragments to single strands in alkali

C) The bands are transferred to a nitrocellulose or nylon membrane by blotting

D) Detection of the DNA fragments on the membrane by using a specific DNA probe

Wells to load samples

Nitrocellulosemembrane

Agarose gel

DNA fragmentcomplementaryto labeled probe

Moving

direction

Transfer buffer

Filter paper sheet

Paper towels

Agarose gel

Single-band imageindicating the positionof the DNA fragment

Nitrocellulosemembrane

3) Northern blotting: the process is similar to that of Southern blotting, except that the sample being analyzed is RNA instead of DNA. It detects RNA molecules using a complementary DNA probe.

2. DNA cloning: a method to prepare large amounts of particular DNA fragments in pure form using DNA recombination technique.

1) Cloning vectors: the most common vectors are bacteriophages and plasmids.

Bacteriophage: also called “phage”, a type of simple microorganism consisting of its own DNA genome in the core and a protein coat outside. It infects bacteria by injecting its DNA into the bacterial cells.

Bacterial host cell

Bacteriophage

Bacterialchromosome

Phage DNA

Bacterial chromosomecarrying phage’s genes

Lysogenic pathway of phage infection

Plasmid: is a small circular double-stranded DNA molecule existing free inside bacterial cells. A plasmid is a mobile genetic element capable of self-replication, and often carries genes for drug resistance, toxins, or breakdown of natural products.

Electron micrograph of phage

2) The basics of DNA cloning: to clone a foreign DNA fragment into a vector DNA, both DNA molecules are cut with restriction enzyme(s), and the two DNA molecules are mixed and are joined covalently by DNA ligase. The recombinant DNA is then introduced into bacterial cells and the recombinant plasmids will be replicated many times. The foreign DNA fragment can be prepared in large amounts or allowed to be expressed in the bacteria.

3) DNA libraries: a DNA library is a collection of cloned DNA fragments in a cloning vector that can be searched for a DNA of interest.

Genomic DNA libraries: made from the genomic DNA of an organism.

Genomic DNA digestion with restriction enzyme DNA fragments cloned into vector selection of the positive colonies (genomic DNA clones)

4) cDNA libraries: made from the reverse transcripts using mRNA as templates. The single stranded cDNA is converted into double stranded DNA and then cloned into a vector (cDNA clone).

A cDNA library contains only the genes for mRNA molecules.

5) Screening DNA libraries: to screen a DNA library for a particular gene using a DNA probe.

Screening genomic libraries:

agar plate with colonies replica of the plate colony pattern on a nitrocellulose membrane detection of the colonies of interest with a DNA probe.

P l a t e w i t h c o l o n i e s

R e p l i c a o f t h e p l a t e

D e t e c t i o n f o r t h ec o l o n i e s o f i n t e r e s t

Screening a gene library

Screening cDNA libraries: similar to that for genomic libraries. In addition to using a DNA probe for selection of the positive colonies, an expression vector can be used to detect the colonies with the target gene.

agar plate with colonies replica on a nitrocellulose membrane detection of the colonies of interest with an antibody against the target protein.

3. Polymerase chain reaction (PCR): a method to make a large number of copies of a given DNA sequence.

1) The principles: a double-stranded DNA molecule is used as template, two primers select the starting sites for DNA fragment synthesis on the two single-stranded DNA molecules. The products are then used as templates to further amplify the synthesis of the target DNA sequences.

2) The reaction steps:A) Denaturation: the template DNA is heated to

95ºC to denature it into single strands;B) Primer annealing: the temperature is lowered

to allow the two primers to bind to the starting sites for DNA synthesis. Generally, the temperature is calculated as: primer Tm-5 =4(G+C)+2(A+T)-5 ;

C) Elongation: the temperature is raised to 72ºC to allow DNA polymerase to elongate each primer using the single stranded DNA templates.

The three steps of PCR are called a cycle, which can be repeated for many times. The copies of the synthesized DNA sequences can be calculated as: 2n, where n is the number of reaction cycles.

e.g. after 20 cycles the original DNA has been amplified a million-fold (220), and 30 cycles can reach a billion-fold (230).

Aplications of PCR: To amplify a single DNA molecule from a

mixture without isolation and purification; To make DNA sequences for sequencing; To change the gene structure such as point

mutation, deletion, and insertion; To detect the existence of microorganisms a

t very small amount levels; To screen for genetic diseases; To provide with genetic evidences in forens

ic medicine.