ch 13 plus whateveah Flashcards
(111 cards)
1
Q
biochemgel electrophoresis
A
analyze, Identify, purify
separates based on size (long at top), charge, or conformation
DNA, RNA, proteins
2
Q
agarose gel electrophoresis used for
A
nucleic acids- bad resolution- horizontal gel
3
Q
pulsed field gel electrophoresis
A
pulses electric charge, for larger fragments of DNA
4
Q
polyacrylamide gel electrophoresis
A
more precises
nucleic acid and proteins
vertical gel
5
Q
how is DNA sorted in electrophoresis
A
big thing at top (near well and negative charge) smaller thing at bottom
6
Q
ethidium bromide
A
glows and shit ehehehehhee
can purify DNA after sorting
7
Q
southern blotting and probe
A
detect DNA, ran on gel electrophoresis and transfer to membrane
Probe is single stranded complementary DNA or
RNA fragment
8
Q
northern blotting
A
detect RNA, like southern
Probe is single stranded complementary DNA or
RNA fragment
9
Q
western blotting
A
detect protein, need to denature ts first, Primary antibody to specific polypeptide,
Secondary antibody to detect/amplify primary, acrylamide
10
Q
probe
A
nucleic acid that has same or similar sequence to gene of interest, labeled and shit,
hybridize with the target
11
Q
antibody
A
detect antigen
12
Q
antibody composition
A
2 heavy chains, 2 light chains, antigen binding site
13
Q
polyclonal antibody is obtained from
A
injecting an animal with the antigen and bleeding it
14
Q
secondary antibodies
A
target fc fragment (constant region)
15
Q
PCR
A
amplifies specific sequence of DNA
16
Q
needed for PCR
A
DNA poly, DNA template, DNA primer, dNTP, buffer/cofacters
17
Q
main polymerase used for PCR
A
Taq DNA poly for thermus aquaticus
18
Q
PCR steps
A
Cycle: Denature, Anneal, and Elongation
19
Q
PCR can be used to identify
A
trinucleotide repeats
20
Q
PCR uses
A
genotyping, mutation detection, forensics, paternity testing, ancestry, trace disease, sequencing, cloning
21
Q
RT-PCR
A
evil RNA PCR
22
Q
RT-PCR great for
A
single/few gene, check transcription levels, test for RNA viruses
23
Q
manual sequencesing/ Sanger is how logn and requires what gel
A
Dna syntehsis reaction, short (500bp), need polyacrlyamide gel
24
Q
Sanger sequencing needs
A
DNA poly
DNA template
free 3’ OH
dNTPs
ddNTPs
primer
25
Sanger steps
elongation, random termination, sorting via gel elctrophoresis, shows complement
26
why is ddNTP a replication terminator
no have 3' OH
27
next gen sequencing
sequining of spatially seperated, clonal amplified DNA templates in array at same time,
for whole genomes
28
automated DNA sequencing
ddNTP is tagged with diff colors, loaded in capillary array to sort, laser beams, electro-gram converted to sequence using computer
29
illumina sequencing
1. amply- bridge PCR
2. sequences- fluorescence signal made by reversible terminators
3. analyze
30
RNA sequencing is for
gene expression, changes in whole genome expression
31
recombinat DNA tech and products
combines DNA form different sources, makes vectors/plasmids
32
molecular cloning is selective - of a
selective amplification of a particular gene or DNA segment
33
transgenic organisms
genome altered with frogmen DNA sequences by artificial means- shows where genes are expressed and expresses recombinant protiens
34
why clone a gene?
amply DNA
DNa sequencing
create gene alteration
study mutations
1st step in making transgenic organisms
35
(5)steps to molecular cloning
1. obtain DNA segment
2. select small molecule of DNA that can self replicate
3. join DNA
4. put into something
5. Id host cells that have this DNA
36
enzymes needed for recombinant DNA
ligase, restriction endonuclease
37
DNA ligase
joins two pieces of DNA by forming phosphodiester bonds
38
Restriction endonuclease
recognized a sequence and cuts at a recognition site
39
restriction endonuclease original purpose
restrict/ prevent viral infection by degrading invading nucleic acid
40
methylase activity
addition of methyl group to protect these sites in DNA sensitive to attack by restriction endonuclease
41
methylate typical target
adenine methylation to 6 methyl adenine
42
most common restriction endonuclease
type II, we use ts a lot
43
type II endocnulcease
Recognize and cut at recognition site, makes sticky/blunt (straight) ends
44
how to get sequences for amp/manipulate
nonspecific shearing from genomic DNA or cDNA
45
how to shear DNA
sanitation or forcing thru fine gauge needle
46
how to make recombinant DNA molecule
insert gene into vector
47
cloning vectors
must have the ability to replicate, an origin of replication, endonuclease cleavage sites, markers, be easy to manipulate
48
expression vector
has all cloning vector, but has promoter sequence and translation signal
49
most common vector
plasmid/ e coli
50
plasmid
circular DNA molecule that replicate separately from host, 5-400 kb pairs
51
transformation
cell intake rando dna and use it
52
cells that can transform
competent
53
how to make cells chemically competent
heat shock, cacl2
54
positive selectable markers
let cell grow
55
negative cell markers
kill cell
56
screenable marker
visible change in appearance
57
why examine genes
determine unctions, reaction mechanisms, make antibodies, examine protein binding partners, make good products
58
transgenic animals can we used for
protein generating
59
gene pharming
exactly how it sounds
60
GFP
bioluminescent gene
61
GFP helps visualize
where genes are expressed, live cells
62
why make transgenic organisms?
hey have useful traits, for therapeutic value, study gene functions
63
how to make transgenic animal
DNA microinjection, Sperm-mediated DNA transfer, Embryonic stem cell transfer retrovirus mediate gene transfer
64
DNA fingerprinting/typing
Identify individuals by examining their DNA,
Sequence polymorphisms, Minisatellites, short tandem, PCR
65
agrose gel length
100-50,000 bp
66
size of Pulsed field gel electrophoresis:
200-400 Mb
67
size of Polyacrylamide gel electrophoresis:
Less than 500 bp
68
RT-PCR
amplifies RNA, check transcription
level of specific gene, test for RNA virus
69
recombinant steps
1. Obtain the DNA segment
2. Select vector
3. Join two DNA fragments
covalently: restriction enzyme
and DNA ligase
4. transformation
5. Select host cells with recombinant DNA
70
clone
genetic identical to donor
71
Somatic Cell Nuclear
Transfer "dolly" methods
nucleus (DNA) of a somatic cell is transferred into an enucleated metaphase-II oocyte for the generation of a new individual, genetically identical to the somatic cell donor
72
somatic cell problems
- Abnormal offspring is very high (Genes functioning incorrectly, Developmental abnormalities)
– Nuclear reprogramming doesn’t always occur correctly
– Cellular aging
– Improper segregation of chromosomes
73
Why Clone Organisms
Understanding Problems in Developmental
Biology
(DNA differentiation? )
* Cloning transgenic animals
* Cloning prize animals
* Wildlife conservation
* Cloning for stem cells – to treat diseases,
organoids
74
mini satellite
15-50
sub telomeric regions
paternity
75
Short tandem repeats (STR)
2-6
entire genome
forensic DNA
due to slippage in DNA rep
76
How may STR/locci FBI use to ermmm yea
13- its in ermm CODIS yea idk the overall genotype frequency is higher, therefore making the probability of a random match higher as well
77
DNA typing used in
criminal cases, paternity, genetic
relationships, identify human remains
78
Sequence polymorphism
slight sequence differences among individuals
79
Minisatellites or short tandem repeats (STRs)
variable
number of copies of repeat sequences possessed by
many organisms
80
electropherogram
Separate fragments via capillary
electrophoresis and image is an
81
Multiplex PCR
amplification of many targets in one reaction- many labeled primers- DNA detected with laser
82
capillary gel electro
basically gel but in a tube and electropherogramed to look at the UV stuff
83
another analysis methods
mitochondrial and y analysis
84
Mitochondrial DNA analysis
Hundreds of mitochondria, Degrades slower,
– Problems: Mutates, heteroplasmy, and the
statistical approach used sometimes make
interpretations difficult. Only maternal lineage
85
Y analysis
father to son relation
86
Genomics
study of whole sets of genes and their interactions rather than single genes or proteins
87
Transcriptomics
analysis of the RNA transcripts
produced by the genotype at a given time
provides a link between the genome, the proteome,
and the cellular phenotype
88
Proteomics
the determination of the structures and
functions of all of the proteins in an organism
89
Genome
the complete haploid genetic
complement of a typical cell
90
Genomics
Sequencing technology
– Computer technology
91
Genome annotation
Determine the location
and function of genes and other critical
sequences
92
Annotation can describe
-Phenotypic function: effect on whole organism
- Cellular function: metabolic process where gene
product participates and/or interacts with other
gene products in a cell
- Molecular function: precise biochemical activity of
gene product
93
Bioinformatics
a discipline combining
information technology with biotechnology
94
Transcriptome
The entire complement of RNA
transcripts present in a given cell or tissue under
specific conditions
95
Microarrays
analyzes transcriptome, you mix two labeled shits, spot that hoe, image, and analyze
96
proteome
The complement of proteins in cell under certain
conditions/ complement of proteins that can be expressed by a givengenome
97
what does proteome reveal
--Structure
– Posttranslational modifications
– Cellular localization
– Detailed functions
– Interactions
98
Genomic medicine
sequencing to find what allele you have to determine treatment outcome/ possibilities
99
Genome-wide association studies
identify
variants associated with human traits and diseases
– SNPs (single nucleotide polymorphism)
– Structural variants
100
Precision / Personalized medicine
using info form sequencing to determine how to treat an individual
101
steps of cells turning cancerous
Immortalization (crazy dividing), transformation (growth makes it go beyond), metastasis (move thru body)
102
what causes cancer
Group of genetically divers disorders, each
with genetic signature (own set of genes)
Multistep disease: 4-8 genetic changes
required
103
Cancer driver genes
mutate and cause tumor growth
– Inherited
– Acquired: spontaneous or environmentally
inflicted
104
Proto-oncogenes and oncogenes:
inappropriate activation of genes that
normally stimulate growth and play a role in
differentiation and apoptosis (only need 1 mutation!)
105
Tumor suppressor genes:
Loss of function of genes that normally inhibit cell growth and division
– Recessive mutation
– Genetic predisposition
106
tumor supressor examples
Retinoblastoma:
* P53:
107
Retinoblastoma
cell cycle master switch with over 100 different interactions.
– Prevents cells from entering S phase
– Important for differentiation and apoptosis
108
* P53:
Guardian of the genome
– DNA damage control system
– Transcription factor with 100+ targets
– Dominant negative protein
109
Other Factors
* Telomerase activity
* Histone protein modification
* Noncoding RNAs
* Chromosomal rearrangements
* Viruses – transformation
* Chemicals: genotoxic or nongenotoxicio.//
110
somatic gene therapy
alters gene expression in non sex cells
used viruses, liposome and naked DNA
111
germline gene therapy
alters germline expression (inherited)
