Biotechnology Flashcards
recombinant DNA technology
artificial modification of DNA, resulting in the production of recombinant DNA
biotechnology
use of biological processes to produce useful products
recombinant DNA
DNA made by inserting the genes from one source into the DNA molecule of a different source
transgenic organisms
organism which has DNA from another species introduced to its own DNA artificially
restriction enzymes
enzymes able to cut strands of DNA at a specific sequence of nucleotides called a recognition site
recognition site
the specific sequence of 4-8 nucleotides at which an enzyme can cut a strand of DNA
straight cut and blunt end
SC: restriction enzyme makes a clean break across 2 strands of DNA, producing blunt ends
BE: both strands of DNA cut such that ends terminate at the base pair
staggered cut and sticky ends
SC: cut produced when restriction enzymes produce fragments of DNA called sticky ends
SE: stretch of unpaired nucleotides in the DNA molecule that overhand at the break in strands
significance of sticky ends
- able to combine with sections of DNA with a complementary ending
- allowing single stranded overhang from 1 DNA fragment to be paired with any other DNA fragment that has a corresponding sequence
naming restriction enzyme
first: genus
2nd 2: strain
roman: when the enzyme was isolated
Ligase what and how
- in double stranded DNA, joins short fragments of single strands of DNA to form a continuous single strand of DNA
- through phosphodiester bonds joining phosphate group to sugar molecule
-after H bonds formed
vector
bacterial plasmid, viral phage, or other agent used to transfer genetic material from one cell to another
plasmids
- small circular strand of DNA distinct from main bacterial genome
- composed of only a few genes
- replicate independently of cell
- Isolating gene of interest
- identify the desired gene
- use restriction enzyme to cut the DNA on either side of the gene
- Adding gene to vector
- use the same restriction enzyme to cut the DNA of the vector
- add the desired gene to the vector
- use DNA ligase to join the 2 sections of DNA
- getting vector into organism
- cloning of the vectors occurs so there are many copies of DNA available to insert into the host cells
- vector introduced into the host cells
- host cells produce the foreign protein using instructions of the gene in the recombinant DNA
examples of recombinant DNA tecnology
- diagnosis and treatment of genetic disorders
- manufacture of large quantities of pure protein for medical products (insulin, growth hormone)
- recombinant vaccines
- DNA vaccines
define PCR and state its 3 steps
allows a small quantity of DNA to be amplified quickly to produce a testable amount
denaturing, annealing, extension
describe denaturation
-high temperature of 96°C is applied to DNA
- breaks hydrogen bonds between the 2 strands of DNA, forming single strands of DNA
describe annealing
-temperature lowered to 54°C
- DNA primers are added and allowed to bind to the single strands of DNA
- each DNA primer is complementary to either end of the section of DNA about to be copied
describe extenstion
- DNA polymerase and nucleotides added to PCR sample
- heated to 72°C
-adds complementary bases to the DNA sections originating with the primers
-extends nucleotide chain, forming a new strand of DNA
wrap up PCR
- Thermocycling: process of repeated heating and cooling, sequence is repeated 20-30 times
- amplifies the amount of DNA created
why does PCR create the not full length of original DNA
- adding bases starts at the primer and not the end of the DNA
- majority of DNA strands have a length between 2 primers, BUT all strands contain DNA region of interest
Why is hot springs bacteria important in PCR?
- provides Taq polymerase for PCR
- does not denature at high temperatures
- able to add complementary bases to the sections of DNA originating with primers without denaturing
- PCR sample can be heated and cooled alternately, DNA polymerase does not need to be replaced at the end of each cycle
define DNA sequencing
determination of the precise order of nucleotides in a sample of DNA, most frequently Sanger method used
4 uses of DNA sequencing
- identify mutations, compare DNA from different organisms
- identify inherited disorders
- maternity or paternity tests
- compare species to track evolutionary changes
Purpose of gel electrophoresis
separates DNA strands based on their lengths, used to compare DNA sequences or DNA profiles
gel electrophoresis set up
- placed in wells of semi-solid gel immersed in a solution of electrolyte
- electrodes present on either ends of the gel, negative electrode closest to DNA, positive electrode on opposite side
how does gel electrophoresis work
- DNA is negatively charged, moves towards positive electrode
- smaller pieces move faster than larger ones, located further away from negative electrode when current is stopped
- forms DNA profile
define DNA profile
banding patterns of DNA fragments unique to an individual formed after being separated by gel electrophoresis,
how to know lengths of DNA strands in the sample after gel electrophoresis?
- DNA ladder : contains segments of DNA with known lengths
- Gel electrophoresis done on both a DNA ladder and a sample at the same time
- results from sample compared to ladder to determine lengths of DNA strands in the sample
state 3 methods to visualise DNA that has been separated
Ethidium bromide, Methylene blue, DNA probes
describe Ethidium bromide
- added to the agar prior to the gel being set
- DNA moves through gel and picks up some of the chemical
- special UV light shone over gel, DNA fluoresce
Describe methylene blue
- dye that binds to DNA when gel is soaked in it
- areas containing DNA stain a deeper blue, visible to the naked eye
Describe DNA probes
- short sections of a single strand of DNA with a radioactive or fluorescent molecule which binds to the DNA being tested
Format for answering GE questions
- use the same restriction enzyme to cut the DNA
- place the samples in wells in the gel close to the negative electrode
- turn the current on
- DNA is negatively charged, moves towards positive electrode
- smaller fragments of DNA travel further than larger fragments
- forming a banding pattern, or the DNA profile of the individual
- DNA samples are then compared, link back to question (Who’s the father?)
