Biotechnology

Restriction Enzymes

cut the desired DNA out of the organism and DNA out of the plasmid
allows for the desired organism DNA to get inserted into the plasmid
cut in any type of DNA as long as it recognizes specific cutting sequences
bacteria makes it to defend against foreign, invading DNA

process of making bacteria to produce human insulin

Restriction enzyme cuts bacterial plasmid and human insulin gene.
Ligase joins cut plasmid and human insulin gene to create recombinant plasmid.
The recombinant plasmid is inserted into a bacterial cell.
The bacterial cell reproduces into cells with the recombinant plasmid that produce insulin.

bacteria and human antigen production

bacteria can be genetically engineered to produce a recombinant plasmid that codes for proteins to fight the antigens

Recombinant organisms and the environment

could disrupt the food chain if released out in the wild with no sort of regulation

Cells culture

the process where cells can be gathered from natural locations and grown in lab containers under controlled conditions
given appropriate food and environment to grow
easier to care for bacteria and yeast than plant, insect, or animal cells
harvested and studied after growth

Cloning

the process of creating a clonal population with identical DNA
streak bacteria on agar to generate single colonies each from a single cell

Looking inside cells

to study molecules like DNA and proteins, scientists break open cells and sort contents
purify single protein species from mixture in cell type based on physical and chemical properties

DNA

stores information of the cell -chemical backbone with A, C, G, T bases along its side

DNA Structure

directional sequence (5' to 3') because DNA subunits are joined head-to-tail
hydrogen bonds between complementary base pairs A and T, C and G
strands run in opposite directions
thousands or even millions of base pairs long -double helix or circular DNA

RNA

retrieve and execute DNA functions
shorter than DNA, has ribose sugar and uracil instead of thymine

Proteins

workhorses of the cell
20 different types
sequence determines 3D structure determines function
usually single-function, but also diverse function (enzymes, hormones, regulatory proteins, structural, antibodies)

DNA Replication

mRNA codes gene in transcription
tRNA in protein assembly line reads mRNA by codon in translation
rRNAs to build ribosome scaffolding to build proteins

linear DNA has 3D consequences

Genes

segment within a DNA molecule singled out for copying into RNA to perform a function
some traits governed by single genes and others by multiple genes
vary in length and replication rate
all cells have all DNA but do not express all of DNA

DNA Ligase

enzyme gluing compatible pieces of DNA together
helps to create recombinant DNA
genes can be cut from plants or animals and placed in bacteria

Plasmids

small circular DNAs in some bacterial cells that replicate on their own
easy to extract and purify and join to foreign DNAs cut with the same enzyme
transformation puts hybrid DNA back in the bacteria
can carry antibiotic genes that allow new bacteria to survive
transformation
the process that introduces hybrid DNA into bacterial cells -now DNA can be perpetuated within the cell

cloning vehicle/cloning vector

the plasmid which carried the foreign DNA

DNA Libraries

collections of cloned sequences -individual DNAs of interest can be fished out with screening
cloning can produce hybrid DNAs

1st step in producing genetically engineered bacteria

use restriction enzyme to cut the gene from human DNA

genetic modification

a cell takes DNA from another source

Gel electrophoresis

helps to compare DNA samples from other sources
separates negatively charged (because of phosphate) DNA fragments in agarose gel
faster ones move farther down
proteins in polyacrylamide split between positive and negative charge and identified by staining
knowing organism's DNA sequence
allows the researcher to study specific genes

genetically modified bacterium

plasmid
foreign gene
recombinant DNA

Identical pattern on gel electrophoresis

same amount of DNA in both samples
fragments of the same size
same DNA molecules

genetically engineered bacteria advantages

genetically engineered bacteria can mass produce pure human proteins

Polymerase Chain Reaction (PCR)

replication of small necessary segment of DNA in a test tube
follows rules of base pairing
used in nearly every experiment or test involving DNA
developed in 1890s and won its inventor a Nobel Prize
each cycle ends with twice the amount of original DNA

Template DNA

starting DNA from dead organism, alive organism or crime scene
different in every experiment
target sequence isolated from this to be replicated several times

DNA Polymerase

enzyme that reads a strand of DNA and makes a complementary copy following base-pairing rules
Taq polymerase used in PCR because it is thermophilic and stable at high temperatures

Primers

short, single-stranded pieces of synthetic DNA binding to complementary regions in template DNA
forward primer and reverse primer to bind to each end in PCR to find needle in the haystack

dNTPs

a mix of all 4 nucleotide building blocks used to build a new strand of DNA

Buffer

solution maintaining a stable pH containing ions enabling polymerase function

PCR steps

Denaturation
Annealing
Extension

Denaturation

samples heated to around 95° C to separate the DNA strands hydrogen bonds -separation of its structure

Annealing

sample is cooled to 50°C - 65° C
precise temperature based on primer sequences used
complementary strands of nucleotides bond with hydrogen bonds
creates short double-stranded DNA segments flanking the target sequence now primed to be copied

Extension

reaction mixture heated to around 72° C to allow polymerase to copy the DNA
optimal function range of Taq polymerase
Taq polymerase adds nucleotides starting from the primers

PCR Product

result of one or many PCR cycles
one cycle yields twice the original number of DNA strands

Taq polymerase

thermophilic enzyme used in PCR for extension step

Thermocycler

a mechanism that is used for PCR that raises the temperature around the DNA to the correct temperature for each of the steps to occur

Sanger Method

developed by Fred Sanger
2 strands of DNA are separated
sequenced strand is copied using chemically altered bases
stops each time one particular letter is found in the chain
process repeats for each letter and the pieces are put together like a jigsaw puzzle to find the original DNA strand

Genetically Modified Organisms (GMOs)

crops that carry new traits that have been inserted through advanced genetic engineering methods

Short-tandem Repeats (STRs)

sections of a chromosome in which DNA sequences are repeated
very short-sequence that is repeated can help identify species -number of repeats that occurs can help identify a person

Somatic cell cloning

commonly referred to as cloning, an embryo developed from the genome of a somatic cell

Cultured adult cells taken from organisms
UV destroys egg's nucleus and adult cell's nucleus placed in the cell
electric shock to stimulate the cell and it develops into a clone

DNA Profiling

technique by which individuals can be identified and compared via their respective DNA profiles using different numbers of STRs at certain loci in satellite DNA in non-coding regions
used in forensic/criminal investigations and paternity testing

Bt corn crops and monarch butterflies

Dr. John Losey studied this
in his 1st experiment, he sprinkled Bt pollen and normal pollen on some leaves and not on others to determine their effects
found that Bt pollen was hurting the butterflies
studied natural conditions for more reliable results and found that it did not
most likely to harm larvae as they feed on milkweed the most