Section 6.1Page 278

BIOTECHNOLOGICAL TOOLS & TECHNIQUES

What is biotechnology?Applied biology

genetics; molecular biology; microbiology; biochemistry

Uses living organisms and their components to create “bio-products”industry, agriculture, medicine

Involves manipulation of DNA

Manipulating DNARecombinant DNA – a fragment of DNA

composed of sequences from at least two different sources

Biotechnological tools and techniques1. Restriction endonucleases2. Methylases3. Ligase4. Gel electrophoresis

Imagine joining two DNA sequences:

You would need tools:Scissors to cut the fragments out of their sourcesGlue to join the fragments together

Biotechnology uses tools that are already existing within biological systems

Restriction endonucleases (RE)aka restriction enzymes“molecular scissors”

What do they do?recognize specific base-pair sequences in DNA, and then

cut the double-stranded DNA at those sites

http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

Function: Crude immune system in bacteriaCleaves virus DNA into fragment

Host DNA is methylated – RE knows not to cleave it

Recognition siteRecognition site: the sequence recognized by the

enzyme

Characteristics:Specific to each different RE (there are over 2500)4-8 bp in lengthUsually palindromic

Ends produced by RE cleavageSticky ends: Cleavage produces an

overhang Depending on where the RE cuts, it an

be a 5’ or a 3’ overhang

Blunt ends: No overhang

For biotechnology, sticky ends are more useful

If two fragments are cut with the same RE, they will have complementary sticky ends

These fragments can be joined (“glued” together)

Naming restriction enzymes

B genus Bacillusam species amyloliquefaciensH strainI first endonuclease isolated from this strain

BamHI

H genus Haemophilusin species influenzaed strain RdII second endonuclease isolated from this strain

HindII

Page 281 practice

SmaI recognition sequence: CCCGGGCuts between the C and the G

Location of cuts?How many fragments?What type of ends?

5’-AATTCGCCCGGGATATTACGGATTATGCATTATCCGCCCGGGATATTTTAGCA-3’3’-TTAAGCGGGCCCTATAATGCCTAATACGTAATAGGCGGGCCCTATAAAATCGT-5’

HindIII recognition sequence: AAGCTTCleaves between the two A’s

What type of ends are produced?

5’-AAGCTT-3’

MethylasesMethylases are enzymesAdd a methyl group to

the recognition sitePrevents RE from cleaving the DNA

Function: Protect host DNA from own RE’sAs a biotechnological tool:

Allow protection of fragments/specific sequences

LigasesWhere have you seen this enzyme before??

DNA replication Joins sugar/phosphate backbones of DNA fragments

Can be used to join fragments that have complementary endsPhosphodiester bond

Most frequently used: T4 DNA ligase

Overview: Producing recombinant DNA

So what enzymes act as the scissors and glue???

Gel electrophoresisMethod of separating DNA fragments

Used in genetic engineering to isolate desired fragments

RE may cut at several sites.

Want to make sure the correct fragment is isolated.

Useful properties of DNA1. DNA is negatively-charged (phosphate

groups)

2. Charge-to-mass ratio of all nucleotides is consistent

Principle of electrophoresisSeparates DNA fragments based on their sizes

Involves forcing DNA fragments through a gel matrix

Matrix acts like a sieve – has pores through which DNA can travel

Separation of fragmentsFragments will migrate through the gel at a rate that is

inversely proportional to logarithm of their size

Smaller fragments will migrate fasterLarger fragments will migrate more slowly

Animation: http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

Procedure1. DNA is cleaved into smaller fragments. Depending

on the cut sites, the fragments will be different sizes.

2. The sample of DNA is loaded into small wells within the gel matrix.

3. A charge is applied across the gel: Negative at the sample end; positive at the opposite.

4. DNA fragments will migrate towards positive pole.

Depending on fragment size, migration rates will vary

Wells/indents within gel

Sizing the bandsA “ladder” of fragments of known sizes is run

alongside samplesCompare samples to bands of known size

Visualizing the DNAStain with ethidium bromideEthidium bromide inserts itself into the DNA

backboneFluoresces under UV light

Obtaining the desired fragmentLiterally cut the band out of the gel

Purify to obtain the fragment

HomeworkPg. 282 #9, 10Pg. 284 #11-14Pg. 291 #2, 3, 6, 8, 14-17