Practical Molecular

Question 1

Human Genome Project

Answer 1

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%)

Question 2

Restriction Endonucleases

Answer 2

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

Question 3

Agarose gel

Answer 3

use in DNA analysis

molecules are already charge, migrate to the positive electrode on their own during electrophoresis

Question 4

SDS page

Answer 4

protein analysis

adds a universal negative charge to the electrode to cause it to move

large, hydrophobic with single negative charge

unfolds protein

Question 5

what do you use if you need to visualize DNA

Answer 5

gels

Agarose or SDS page

Question 6

what do you use if you want to move or manipulate DNA to create a gene library?

Answer 6

restriction endonuclease

Question 7

which gel would you use in DNA analysis?

Answer 7

agarose gel

SDS is protein analysis

Question 8

what is utilized to move DNA in a gel?

Answer 8

bacteria

Question 9

how are bacteria used in a gel?

Answer 9

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

Question 10

ligase

Answer 10

glues fragments of RE together in DNA

Question 11

what is a DNA library?

Answer 11

genes are put into bacteria and stored

two types: cDNA, genomic

Question 12

cDNA library

Answer 12

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

Question 13

genomic DNA

Answer 13

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

Question 14

FISH

Answer 14

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

Question 15

PCR

Answer 15

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)

Question 16

STR

Answer 16

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

Question 17

3 types of cell cultures

Answer 17

primary cell cultures

continuous cell line

immortalized cell line

Question 18

primary cell culture

Answer 18

derrived directly from tissue, grow on plastic for limited time

survive for FINITE period of time (scenesence)

isolate cells from heterogenous population

Question 19

continuous cell line

Answer 19

opportunistic (deprived from cancer)

immortalized

from homogenous cell population

more differentiated phenotype

infinite life span in vitro

Question 20

what is in between primary or continuous cell lines?

Answer 20

immortalized cell line

Question 21

immortalized cell line

Answer 21

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

Question 22

morphologies of cell line

Answer 22

fibroblastic
epithelial like
lymphoblast like

Question 23

fibroblastic

Answer 23

bipolar/multipolar, have elongated shapes, grow attached to substrate

i.e. mesenchymal

Question 24

epithelial like

Answer 24

polygonal shape

more rectangular dimension, grow attached to substrate in patches

i.e. Skin cells

Question 25

lymphoblast like

Answer 25

spherical shape

grown in suspension without attaching to surface

e.x. blood biology

Question 26

2D gels and SDS page

Answer 26

able to identify SLIGHT changes in genomic DNA

allows you to resolve post translational modifications

change the isoelectric focusing point (pH)

after analysis of the gel, if there are different locations for the protein it is change

req. known protein– know that they’re different

Question 27

what do you use for identifying an unknown protein?

Answer 27

mass spectrometry

Question 28

western blotting

Answer 28

use for known protein

Ab associates directly with immobilized protein from SDS page gel transferred to blow

tells you amount of what you have

Question 29

immunoprecipitation

Answer 29

use to isolate one protein from mixture of protein

can use to find unknown interacting partners

just use for e-cadherin and then resolve on gel

Question 30

ELISA

Answer 30

MOST clinically relevant, like western blot

either looking for protein of interest and have Ab, (present and quantified) or use to quantify the amount of an Ab in blood

similar to western blot but doesn’t separate samples

look for protein of interest with Ab specific for it

one it BINDS to Ab, sends signal using another Ab

Question 31

types of ELISA

Answer 31

direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

Question 32

sandwich ELISA

Answer 32

Ab already immobilized, binds to another Ab then binds to complex and makes color change

ex. pregnancy test

Question 33

which library is a random collection of DNA pieces due to restriction endonuclease activity?

Answer 33

genomic library

Question 34

which library to analyze just one gene?

Answer 34

cDNA library

Question 35

what are the restriction endonuclease

Answer 35

HaeIII
EcoRI
HindIII