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Molecular Genetics > ch 13 plus whateveah > Flashcards

ch 13 plus whateveah Flashcards

1
Q

gel electrophoresis

A

analyze, Identify, purify
separates based on size (long at top), charge, or conformation
DNA, RNA, proteins

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2
Q

agarose gel electrophoresis used for

A

nucleic acids- bad resolution- horizontal gel

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3
Q

pulsed field gel electrophoresis

A

pulses electric charge, for larger fragments of DNA

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4
Q

polyacrylamide gel electrophoresis

A

more precises
nucleic acid and proteins
vertical gel

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5
Q

how is DNA sorted in electrophoresis

A

big thing at top (near well and negative charge) smaller thing at bottom

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6
Q

ethidium bromide

A

glows and shit ehehehehhee
can purify DNA after sorting

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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

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8
Q

northern blotting

A

detect RNA, like southern
Probe is single stranded complementary DNA or
RNA fragment

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9
Q

western blotting

A

detect protein, need to denature ts first, Primary antibody to specific polypeptide,
Secondary antibody to detect/amplify primary, acrylamide

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10
Q

probe

A

nucleic acid that has same or similar sequence to gene of interest, labeled and shit,
hybridize with the target

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11
Q

antibody

A

detect antigen

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12
Q

antibody composition

A

2 heavy chains, 2 light chains, antigen binding site

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13
Q

polyclonal antibody is obtained from

A

injecting an animal with the antigen and bleeding it

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14
Q

secondary antibodies

A

target fc fragment (constant region)

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15
Q

PCR

A

amplifies specific sequence of DNA

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16
Q

needed for PCR

A

DNA poly, DNA template, DNA primer, dNTP, buffer/cofacters

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17
Q

main polymerase used for PCR

A

Taq DNA poly for thermus aquaticus

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18
Q

PCR steps

A

Cycle: Denature, Anneal, and Elongation

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19
Q

PCR can be used to identify

A

trinucleotide repeats

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20
Q

PCR uses

A

genotyping, mutation detection, forensics, paternity testing, ancestry, trace disease, sequencing, cloning

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21
Q

RT-PCR

A

evil RNA PCR

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22
Q

RT-PCR great for

A

single/few gene, check transcription levels, test for RNA viruses

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23
Q

manual sequencesing/ Sanger is how logn and requires what gel

A

Dna syntehsis reaction, short (500bp), need polyacrlyamide gel

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24
Q

Sanger sequencing needs

A

DNA poly
DNA template
free 3’ OH
dNTPs
ddNTPs
primer

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25
Q

Sanger steps

A

elongation, random termination, sorting via gel elctrophoresis, shows complement

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26
Q

why is ddNTP a replication terminator

A

no have 3’ OH

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27
Q

next gen sequencing

A

sequining of spatially seperated, clonal amplified DNA templates in array at same time,
for whole genomes

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28
Q

automated DNA sequencing

A

ddNTP is tagged with diff colors, loaded in capillary array to sort, laser beams, electro-gram converted to sequence using computer

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29
Q

illumina sequencing

A
  1. amply- bridge PCR
  2. sequences- fluorescence signal made by reversible terminators
  3. analyze
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30
Q

RNA sequencing is for

A

gene expression, changes in whole genome expression

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31
Q

recombinat DNA tech and products

A

combines DNA form different sources, makes vectors/plasmids

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32
Q

molecular cloning is selective - of a

A

selective amplification of a particular gene or DNA segment

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33
Q

transgenic organisms

A

genome altered with frogmen DNA sequences by artificial means- shows where genes are expressed and expresses recombinant protiens

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34
Q

why clone a gene?

A

amply DNA
DNa sequencing
create gene alteration
study mutations
1st step in making transgenic organisms

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35
Q

(5)steps to molecular cloning

A
  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
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36
Q

enzymes needed for recombinant DNA

A

ligase, restriction endonuclease

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37
Q

DNA ligase

A

joins two pieces of DNA by forming phosphodiester bonds

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38
Q

Restriction endonuclease

A

recognized a sequence and cuts at a recognition site

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39
Q

restriction endonuclease original purpose

A

restrict/ prevent viral infection by degrading invading nucleic acid

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40
Q

methylase activity

A

addition of methyl group to protect these sites in DNA sensitive to attack by restriction endonuclease

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41
Q

methylate typical target

A

adenine methylation to 6 methyl adenine

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42
Q

most common restriction endonuclease

A

type II, we use ts a lot

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43
Q

type II endocnulcease

A

Recognize and cut at recognition site, makes sticky/blunt (straight) ends

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44
Q

how to get sequences for amp/manipulate

A

nonspecific shearing from genomic DNA or cDNA

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45
Q

how to shear DNA

A

sanitation or forcing thru fine gauge needle

46
Q

how to make recombinant DNA molecule

A

insert gene into vector

47
Q

cloning vectors

A

must have the ability to replicate, an origin of replication, endonuclease cleavage sites, markers, be easy to manipulate

48
Q

expression vector

A

has all cloning vector, but has promoter sequence and translation signal

49
Q

most common vector

A

plasmid/ e coli

50
Q

plasmid

A

circular DNA molecule that replicate separately from host, 5-400 kb pairs

51
Q

transformation

A

cell intake rando dna and use it

52
Q

cells that can transform

A

competent

53
Q

how to make cells chemically competent

A

heat shock, cacl2

54
Q

positive selectable markers

A

let cell grow

55
Q

negative cell markers

A

kill cell

56
Q

screenable marker

A

visible change in appearance

57
Q

why examine genes

A

determine unctions, reaction mechanisms, make antibodies, examine protein binding partners, make good products

58
Q

transgenic animals can we used for

A

protein generating

59
Q

gene pharming

A

exactly how it sounds

60
Q

GFP

A

bioluminescent gene

61
Q

GFP helps visualize

A

where genes are expressed, live cells

62
Q

why make transgenic organisms?

A

hey have useful traits, for therapeutic value, study gene functions

63
Q

how to make transgenic animal

A

DNA microinjection, Sperm-mediated DNA transfer, Embryonic stem cell transfer retrovirus mediate gene transfer

64
Q

DNA fingerprinting/typing

A

Identify individuals by examining their DNA,
Sequence polymorphisms, Minisatellites, short tandem, PCR

65
Q

agrose gel length

A

100-50,000 bp

66
Q

size of Pulsed field gel electrophoresis:

A

200-400 Mb

67
Q

size of Polyacrylamide gel electrophoresis:

A

Less than 500 bp

68
Q

RT-PCR

A

amplifies RNA, check transcription
level of specific gene, test for RNA virus

69
Q

recombinant steps

A
  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
Q

clone

A

genetic identical to donor

71
Q

Somatic Cell Nuclear
Transfer “dolly” methods

A

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
Q

somatic cell problems

A
  • Abnormal offspring is very high (Genes functioning incorrectly, Developmental abnormalities)
    – Nuclear reprogramming doesn’t always occur correctly
    – Cellular aging
    – Improper segregation of chromosomes
73
Q

Why Clone Organisms

A

Understanding Problems in Developmental
Biology
(DNA differentiation? )
* Cloning transgenic animals
* Cloning prize animals
* Wildlife conservation
* Cloning for stem cells – to treat diseases,
organoids

74
Q

mini satellite

A

15-50
sub telomeric regions
paternity

75
Q

Short tandem repeats (STR)

A

2-6
entire genome
forensic DNA
due to slippage in DNA rep

76
Q

How may STR/locci FBI use to ermmm yea

A

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
Q

DNA typing used in

A

criminal cases, paternity, genetic
relationships, identify human remains

78
Q

Sequence polymorphism

A

slight sequence differences among individuals

79
Q

Minisatellites or short tandem repeats (STRs)

A

variable
number of copies of repeat sequences possessed by
many organisms

80
Q

electropherogram

A

Separate fragments via capillary
electrophoresis and image is an

81
Q

Multiplex PCR

A

amplification of many targets in one reaction- many labeled primers- DNA detected with laser

82
Q

capillary gel electro

A

basically gel but in a tube and electropherogramed to look at the UV stuff

83
Q

another analysis methods

A

mitochondrial and y analysis

84
Q

Mitochondrial DNA analysis

A

Hundreds of mitochondria, Degrades slower,
– Problems: Mutates, heteroplasmy, and the
statistical approach used sometimes make
interpretations difficult. Only maternal lineage

85
Q

Y analysis

A

father to son relation

86
Q

Genomics

A

study of whole sets of genes and their interactions rather than single genes or proteins

87
Q

Transcriptomics

A

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
Q

Proteomics

A

the determination of the structures and
functions of all of the proteins in an organism

89
Q

Genome

A

the complete haploid genetic
complement of a typical cell

90
Q

Genomics

A

Sequencing technology
– Computer technology

91
Q

Genome annotation

A

Determine the location
and function of genes and other critical
sequences

92
Q

Annotation can describe

A

-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
Q

Bioinformatics

A

a discipline combining
information technology with biotechnology

94
Q

Transcriptome

A

The entire complement of RNA
transcripts present in a given cell or tissue under
specific conditions

95
Q

Microarrays

A

analyzes transcriptome, you mix two labeled shits, spot that hoe, image, and analyze

96
Q

proteome

A

The complement of proteins in cell under certain
conditions/ complement of proteins that can be expressed by a givengenome

97
Q

what does proteome reveal

A

–Structure
– Posttranslational modifications
– Cellular localization
– Detailed functions
– Interactions

98
Q

Genomic medicine

A

sequencing to find what allele you have to determine treatment outcome/ possibilities

99
Q

Genome-wide association studies

A

identify
variants associated with human traits and diseases
– SNPs (single nucleotide polymorphism)
– Structural variants

100
Q

Precision / Personalized medicine

A

using info form sequencing to determine how to treat an individual

101
Q

steps of cells turning cancerous

A

Immortalization (crazy dividing), transformation (growth makes it go beyond), metastasis (move thru body)

102
Q

what causes cancer

A

Group of genetically divers disorders, each
with genetic signature (own set of genes)
Multistep disease: 4-8 genetic changes
required

103
Q

Cancer driver genes

A

mutate and cause tumor growth
– Inherited
– Acquired: spontaneous or environmentally
inflicted

104
Q

Proto-oncogenes and oncogenes:

A

inappropriate activation of genes that
normally stimulate growth and play a role in
differentiation and apoptosis (only need 1 mutation!)

105
Q

Tumor suppressor genes:

A

Loss of function of genes that normally inhibit cell growth and division
– Recessive mutation
– Genetic predisposition

106
Q

tumor supressor examples

A

Retinoblastoma:
* P53:

107
Q

Retinoblastoma

A

cell cycle master switch with over 100 different interactions.
– Prevents cells from entering S phase
– Important for differentiation and apoptosis

108
Q
  • P53:
A

Guardian of the genome
– DNA damage control system
– Transcription factor with 100+ targets
– Dominant negative protein

109
Q

Other Factors

A
  • Telomerase activity
  • Histone protein modification
  • Noncoding RNAs
  • Chromosomal rearrangements
  • Viruses – transformation
  • Chemicals: genotoxic or nongenotoxicio.//
110
Q

somatic gene therapy

A

alters gene expression in non sex cells
used viruses, liposome and naked DNA

111
Q

germline gene therapy

A

alters germline expression (inherited)

Molecular Genetics (13 decks)

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