TOOLS OF GENETIC ENGINEERING

There are a number of tools used in genetic engineering. One of them are enzymes.

ENZYMES

There are a number of enzymes used in genetic engineering as important biological tools. Some of them are discussed.

ENZYMES USED IN GENETIC ENGINEERING

Restriction Endonucleases. DNA Ligase. Si- Nuclease. Reverse Transcriptase. DNA Polymerase. Alkaline Phosphatase.

ENZYMES USED IN GENETIC ENGINEERING RESTRICTION ENDONUCLEASE

RESTRICTION ENDONUCLEASES

Called “Restriction Enzymes” because they restrict host range for certain bacteriophage. Called “Restriction Enzymes” because they restrict host range for certain bacteriophage. Bacterial" immune system": destroy any "non-self" DNA. Bacterial" immune system": destroy any "non-self" DNA.

RESTRICTION ENDONUCLEASES Methylase recognizes same sequence in host DNA and protects it by methylating it. Restriction Enzyme destroys unprotected = non-self DNA (restriction/modification systems).

RESTRICTION ENDONUCLEASES  Also called restriction enzymes.  Occur naturally in bacteria.  Hundreds are purified and available commercially.

RESTRICTION ENDONUCLEASES  Named for bacterial genus, species, strain and type.  Example:EcoRI  Genus: Escherichia  Species: coli  Strain: R  Type: I

RESTRICTION ENDONUCLEASES  Recognize specific base sequences in DNA.  Cut DNA at those recognition sites.

RESTRICTION ENDONUCLEASES Restriction Sites:  Enzymes recognize specific 4-8 bp sequences EcoRI5’…GAATTC…3’ 3’…CTTAAG…5’

RESTRICTION ENDONUCLEASES Restriction Sites:  Recognition sites have symmetry.  Some enzymes cut in a staggered fashion.  Some enzymes cut in a direct fashion. PvuII5’…CAGCTG…3’ 3’…GTCGAC…5’

RESTRICTION ENDONUCLEASES Products generated by Restriction Enzymes: COHESIVE END CUTTERS (staggered cuts): Enzyme Recognition Site Ends of DNA After Cut EcoRI5’…GAATTC…3’5’…GAATTC…3’ 3’…CTTAAG…5’3’…CTTAA G…5’ PstI5’…CTGCAG…3’5’…CTGCA G…3’ 3’…GACGTC…5’3’…GACGTC…5’

RESTRICTION ENDONUCLEASES Products generated by Restriction Enzymes: BLUNT END CUTTERS (direct cuts): Enzyme Recognition Site Ends of DNA After Cut HaeIII5’…GGCC…3’ 5’…GG CC…3’ 3’…CCGG…5’ 3’…CC GG…5’

COMMONLY USED RESTRICTION ENZYMES EcoRI 5'-G | AATTC EcoRV 5'-GAT | ATC HinDIII 5'-A | AGCTT SacI 5'-GAGCT | C

COMMONLY USED RESTRICTION ENZYMES SmaI 5'-CCC | GGG XmaI 5'-C | CCGGG BamHII 5'-G | GATCC PstII 5'-CTGCA | G

FREQUENCY OF CUTTING Average distance between cuts is: 4 n where “n” is number of bp’s in recognition site.

FREQUENCY OF CUTTING 4-base cutter:4 4 = 256 bp 5-base cutter:4 5 = 1,024 bp 6-base cutter:4 6 = 4,096 bp 8-base cutter:4 8 = 65,536 bp

ENVIRONMENTAL CONDITIONS

The activity of restriction enzymes is dependent upon precise environmental conditions:  pH.  Temperature.  Salt Concentration.  Ions.

ENZYME UNIT

ENZYME UNITS  An Enzymatic Unit (u) is defined as the amount of enzyme required to digest 1 ug of DNA under optimal conditions: 3-5 u/ug of genomic DNA. 1 u/ug of plasmid DNA.

TYPES OF RESTRICTION ENZYMES

There are three major types of restriction enzymes They are:  Type I  Type II  Type III

TYPES OF RESTRICTION ENZYMES Type I - Recognize specific sequences and cut DNA at a nonspecific site > than 1,000 bp away. Type II - Recognize palindromic sequences and cut within the palindrome. Type III - Recognize specific 5-7 bp sequences and cut bp down stream of the site.

TYPE II RESTRICTION ENZYMES

Type II restriction endonucleases are the most useful class as they recognize specific palindromic sequences in DNA and cut the sugar phosphate backbone within the palindrome.

PALINDROME

A palindrome is anything that reads the same forwards and backwards. English palindromes: Mom. Dad.

DNA PALINDROME DNA is double stranded and the strands run anti parallel. Palindromes are defined as any double stranded DNA in which reading 5’ to 3’ both are the same.

DNA PALINDROME PvuII5’…CAGCTG…3’ 3’…GTCGAC…5’ PstI5’…CTGCAG…3’ 3’…GACGTC…5’

USES AND APPLICATIONS OF RESTRICTION ENZYMES

Bacterial Immune Systems: Bacterial Immune Systems: Recognition of non-self viral DNA. Literally chopping of viral DNA.

USES AND APPLICATIONS OF RESTRICTION ENZYMES DNA Finger printing: DNA Finger printing: Because restriction endonucleases cut specific sequences they can be used to make “DNA fingerprints” of different samples of DNA. As long as the cutting site changes on the DNA or the distance between cutting sites changes, fragments of different sizes will be made.

USES AND APPLICATIONS OF RESTRICTION ENZYMES DNA Cloning: DNA Cloning: Because Type II restriction endonucleases cut at palindromes, they may leave “sticky ends” that will base pair with any other fragment of DNA cut with the same enzyme. This is useful in cloning.

TOOLS

Different tools are being used for identifying restriction sites within a sequence (either genomic or plasmid). Different tools are being used for identifying restriction sites within a sequence (either genomic or plasmid).

TOOL RESTRICTION MAPPER RESTRICTION MAPPER

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