SBI4U: Biotechnology Tools and Techniques

What is biotechnology?Applied biology

genetics; molecular biology; microbiology; biochemistryUses living organisms and their components to create “______________________”

industry, agriculture, medicineInvolves _____________________________

Manipulating DNARecombinant DNA: A Fragment of DNA composed of ___________________________________

Biotechnology Tools and Techniques1. Restriction Endonucleases2. Methylases3. Ligase4. Gel Electrophoresis

To join 2 DNA Sequences you need scissors and glue, but where can we find these items in a cell???

Restriction Endonucleases aka ________________________________ “molecular scissors”

What do they do? recognize ______________________________________, and then ________ the double-

stranded _____________________________

Function: Crude immune system in _______________ Cleaves _____ DNA into fragment Host DNA is methylated:

RE knows not to cleave it

Recognition site: the sequence ____________________________________Characteristics:

Specific to each _______________________________ (there are over 2500) _____________________________ in length Usually palindromic

What is a palindrome?Example 1: MADAM I'M ADAMExample 2: Recognition site of restriction enzyme EcoRI:5’-GAATC -3’3’-CTTAG-5’

Sticky ends: Cleavage produces an ___________ Depending on where the RE cuts,

it can be a ____________________

Blunt ends: _______________________

For biotechnology, _________________________________ If two fragments are cut with the same RE, they will have

____________________________________ These fragments can be joined (“________________________” together)

Practice:

SmaI recognition sequence: CCCGGGCuts between the C and the G

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

HindIII recognition sequence: AAGCTT Cleaves between the two A’s What type of ends are produced?

5’-AAGCTT-3’

Methylases Methylases are ______________ Add a methyl group to

the ___________________ Prevents _________________________ Function: ____________________ from own RE’s As a biotechnological tool:

Allow protection of ____________________ _________________________

Ligases Where have you seen this enzyme before??

________________________ Joins ____________________________

Can be used to join fragments that have __________________________________

_________________________ bond Most frequently used: ____________________

Gel Electrophoresis Method of separating DNA fragments Used in genetic engineering to isolate

____________________________ RE may cut at several sites. Want to make sure the ________________

__________________________________

Useful properties of DNA1. DNA is __________________________

(phosphate groups)2. Charge-to-mass ratio of all nucleotides is

__________________________

Principles of Gel Electorphoresis Separates DNA fragments based __________________ Involves forcing DNA fragments ____________________________________ Matrix acts like a sieve – has pores through which ________________________ Fragments will migrate through the gel at a rate that is ______________________________

to logarithm of their size Smaller fragments will ________________________ Larger fragments will _________________________

Producing Recombinant DNA

Procedure1. DNA is cleaved into ________________________. Depending on the cut sites, the fragments

will be different sizes.2. The sample of DNA is loaded into _____________________ 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 ___________________________________________

Depending on fragment size, migration rates will vary

Sizing the Bands A “____________________” of fragments of known sizes is run alongside samples Compare samples to ___________________________________

Visualizing the DNA Stain with ___________________________ Ethidium bromide inserts itself into the DNA backbone Fluoresces under ______________________

Obtaining the desired fragment Literally __________________________ Purify to obtain the fragment