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BIOTECHNOLOGICAL TOOLS & TECHNIQUES. Section 6.1 Page 278. 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. - PowerPoint PPT Presentation
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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