biotechnology Flashcards
what is bio technology
-The manipulation of living organisms or their components to perform practical tasks or provide useful products
-Advances in biotechnology has improved our agricultural, medical and environmental practices
-Using tech to change genetic information (DNA and RNA)
GMO: genetically modified organisms
A molecular biologist’s toolkit
-Various tools in molecular biology are used to insert foreign genes into other organisms to produce recombinant DNA (a fragment of DNA composed of sequences originating from at least two sources)
These tools include:
Restriction endonucleases (or enzymes)
Vectors (plasmids) - from bacteria
CRISPR-Cas9
Restriction endonucleases/enzymes
Specific enzymes (found in bacteria) that recognize specific short sequences of DNA and cleave the DNA at or near the recognition sequence
Molecular scissors - they can recognize very particular sites
The cutting of DNA using REs is known as a restriction digest
Recognition sequences: usually 4 or 6 bases, but there are some that are 5, 8, or longer
Recognition sequences are palindromes
Palindrome: sequence of DNA that is the same when one strand is read from left to right or the other strand is read from right to left
Restriction enzymes are isolated from bacteria
EcoR1 - E. coli
Sma1 - Serratia marcescens
HindIII - Haemophilus parainfluenzae
BamH1 - Bacillus amyloli
DNA digestion
After a recognition site has been cut on a given fragment of DNA, two types of ends are created:
Sticky ends: short overhanging nucleotides capable of forming H-bonds with other sticky ends (EcoR1)
Blunt ends: fully paired bases at the end of fragment (Sma1), ligase is used to join complementary blunt ends
The ends can be permanently bonded using DNA ligase (phosphodiester bonds reformed)
Vectors
Vectors are carriers of recombinant DNA
Two most popular types: (1) bacterial plasmids (2) viruses
Bacterial DNA cannot be changed (it will change what the bacteria is), plasmids can be changed
A closer look at plasmids
Plasmids are small, circular, double stranded pieces of DNA that can exit and enter bacterial cells
Plasmids require the cellular machinery of bacteria (e.g. ribosomes and enzymes) to replicate and express the genes contained within it
Benefits to bacteria:
Antibiotic resistance: plasmids often carry genes that express proteins that offer resistance to antibiotics
Herbicide resistance: genes that break down herbicides
Plasmids
Restriction endonucleases are used to splice foreign DNA into plasmids
REs cut at the same recognition site, creating complementary “sticky ends” that can anneal to form a “transformed plasmid”
This new plasmid can then be placed in a bacterial cell for replication
CaCl
Electroporation
CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial defense system consists of repeating sequences of genetic code, interrupted by “spacer” sequences - remnants of genetic code from past invaders (bacteriophages)
“Spacer” allows bacteria to recognize and future invasions from the same virus
CRISPR-Cas9 genome editing technology - RNA fragment inserted into “spacer” that recognizes DNA, then Cas9 (enzyme) cuts DNA at specific sequence, shutting off targeted gene
Polymerase Chain Reaction (PCR)
One of the most powerful tools in molecular biology
Invented by Kary Mullis in 1983, resulting in his Nobel Prize in Chemistry
In essence, this process acts as a “copying machine” for DNA
The reaction mixture
Target DNA
Primers
Free nucleotides
Taq DNA polymerase
Buffer containing magnesium
The basic protocol
Denaturation of DNA to single strands
Annealing of primers to DNA
Extension by polymerase
Repeat 30-35 times
What makes it work?
