Practical Molecular Flashcards
Human Genome Project
originally thought there were 100k
26,000 genes to run the human body (as many as plant)
1.5% of the genome codes for genetic expression/exons, rest is “junk DNA” (controls 2%)
Restriction Endonucleases
use if you want to cut DNA/movement of DNA
Manipulation of DNA
cut it apart and glue it back together
manipulate specific genes to generate library
NOTHING with proteins or gels, can’t visualize
Agarose gel
use in DNA analysis
molecules are already charge, migrate to the positive electrode on their own during electrophoresis
SDS page
protein analysis
adds a universal negative charge to the electrode to cause it to move
large, hydrophobic with single negative charge
unfolds protein
what do you use if you need to visualize DNA
gels
Agarose or SDS page
what do you use if you want to move or manipulate DNA to create a gene library?
restriction endonuclease
which gel would you use in DNA analysis?
agarose gel
SDS is protein analysis
what is utilized to move DNA in a gel?
bacteria
how are bacteria used in a gel?
cut bacteria with restriction endonuclease
glue it into circular plasmid
put in plasmid that looks like bacterial genome
ligase glues the ends together to incorporate the plasmid
*MUST be double stranded DNA
ligase
glues fragments of RE together in DNA
what is a DNA library?
genes are put into bacteria and stored
two types: cDNA, genomic
cDNA library
DNA copy of mRNA
generated from the message on the gene
no introns (just extrons)
composed of just start codon and polyadenylation sites –> what feeds into ribosome
requires reverse transcriptase, glued into library
useful for analysis of 1 just gene/protein
genomic DNA
pieces of DNA then glue together to get a library
ea. bacteria only replicates one plasmid
you get a bunch of random fragments due to restriction endonuclease activity that are glued into plasmids to get a library
each plate represents ea. piece of the genome
utilized to sequence entire genome sequence
FISH
use single stranded DNA to bind to an existing DNA in situ
localize gene of interest during an event
analyze presence and location of genes
amniocentesis
PCR
DNA replication, basic foundation of all these tests
utilizes primers (short, complementary strands of DNA)
allows you to amplify long non-coding regions of DNA (STRs)
STR
short tandem repeats
regions between genes, passed down, replicated and maintained
repeated generation after generation
not always part of protein expression (not very regulated)
PCR- used in CSI and paternity tests
3 types of cell cultures
primary cell cultures
continuous cell line
immortalized cell line
primary cell culture
derrived directly from tissue, grow on plastic for limited time
survive for FINITE period of time (scenesence)
isolate cells from heterogenous population
continuous cell line
opportunistic (deprived from cancer)
immortalized
from homogenous cell population
more differentiated phenotype
infinite life span in vitro
what is in between primary or continuous cell lines?
immortalized cell line
immortalized cell line
able to stay continually alive (from primary cell)
act more normal (not cancer) but were tricked into continuous division – limit differentiation properties
considered a cell line because it keeps dividing but came from primary cells
morphologies of cell line
fibroblastic
epithelial like
lymphoblast like
fibroblastic
bipolar/multipolar, have elongated shapes, grow attached to substrate
i.e. mesenchymal
epithelial like
polygonal shape
more rectangular dimension, grow attached to substrate in patches
i.e. Skin cells
