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BCHM 218 > Module 3 > Flashcards

Module 3 Flashcards

1
Q

nucleoside vs nucleotide

A

Nucleoside- nitrogenous base + pentose sugar (adenosine, guanosine, cytidine, thymidine, uridine)
Nucleotide- phosphates + pentose sugar + nitrogenous base

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2
Q

chargaff’s rule

A

importance of DNA to heritability because of the base composition
Genetic base composition does not change just what gets turned off and on differentiates things

# of A=T and C=G to maintain nucleotide order and sum of purines = pyrimidines

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3
Q

Differnce between RNA and DNA- in alkaline conditions

A

dna is resistance to alkaline conditions (basic 7-14) becuse it is lacking the 3’OH (phosphodiester bond breaks from RNA- hydrolyze)

  • dna more stable
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4
Q

how were the dimensions and water content of DNA determined

A

X-ray crystallography by Rosaland franklin

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5
Q

what is one helical turn of DNA

A

10 bp and 34A

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6
Q

main charteristic of RNA

A

transient and extra hydrogen for H-bonds

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7
Q

coding RNA =

A

1.5%

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8
Q

Non-coding RNA =

A

85%

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9
Q

Non-cononical base interactions

A

occur in RNA
- Non-watson crick pairing: A-A or G-U allowing for wobble
- Base-triple interactions: C-G-C can allow for more H-bonds

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10
Q

Hair pin

A
  • most common
    nt arranged to maximize H-bonding and base stacking
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11
Q

internal loop

A

dsRNA separte due to lack of watson crick

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12
Q

DNA stability

A
  1. hydrogen bonding
  2. hydrophobic stacking and vanderwaal forces
  3. Ionic interactions
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13
Q

How does Hydrogen bonding effect DNA stability

A

hydrogenbonding keeps the two strands together
- A-T allows for more compressibility

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14
Q

what disrupts H-bonding

A

organic solvents

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15
Q

How does hydrophobic stacking and vanderwaal forces effect DNA stability

A

keeps the hydrophobic outside and hydrophillic inside

vanderwaals are useful do that if one bond breaks the rest do

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16
Q

what forces cause DNA stacking

A

van der walls

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17
Q

How does ionic interactions effect DNA stability

A

Na+ and Mg++ stabilizes the backbone of DNA
- neutrilize the electrostatic repulsion

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18
Q

extincition coefficient units

A

g/cm/L

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19
Q

how to promote DNA melting
- salt
- temp
- ph

in living cells
- dna binding proteins
- organic solvent s

A

decrease
increase
increase

decrease
increase

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20
Q

hyperchromicity

A

the increase of absorbance
nucleotides -> ssdna -> dsdna

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21
Q

increase in absorbance from ssdna to dsdna

A

40%
dsDNA x 1.4 = ssDNA absorbance

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21
Q

what does ph do to dna

A

high ph causes deprotonation of nt = reduced h bond

increase ph = decrease stability

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22
Q

DNA hybirdization depends on

A

stringency and can form btw DNA-RNA DNA-DNA and RNA-RNA

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23
Q

What is stringency

A

how much of conditions of the experiment allow for imperfect pairing of molecules

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24
Q

low stringency =

A

allow for mismatch pairing becuse the conditions are favorable
increase salt
decrease temp
no os

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25
Q

high stringency=

A

to anneal 100% complementnary the conditions dont have to be favorable
decrase salt
increase temp
yes os

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26
Q

PCR- equelence of in vitro to vivo
vivo= primase, pol 3, helicase

A

dna primers, taq polymerase , heat

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27
Q

PCR steps

A

denaturation, reannealing, elongation, repeat

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28
Q

primer design features
- length, gc conten, Ta, GC residues on 3’

A
  • 18-25 nt
  • 40-60% GC content, more = more stable primer
  • Ta around 50-60 degrees
  • 1-2 residues on 3’ end so that new DNA is tight
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29
Q

polymerase catalyze rate

A

1000 bp/min

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30
Q

equation for PCR product

A

2^x
x= cycles

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31
Q

How to visulize PCR

A

agrose gel electrophorisis
- denaturing solution
- separtion by size- small run faster

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32
Q

How to optimize PCR (stingency + why)

A

increase stringency conditions
so product is more clear
- incresasing annealing temp
- reduce salt

so that the primers dont need to be 100% right
but if to many contaminated products would want to increase stringency

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32
Q

ethimine bromide

A

used to visulize in PCR
- intercalaytes between dna

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33
Q

what is RT-PCR

A

PCR but on RNA and will quantify mRNA levels

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34
Q

What is end point PCR

A

only anazlyse the end product do know know wjat is in each step
- is the dna templatte present

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35
Q

q-PCR

A

real time can quanitfy after each step
- use flouresents
- how much template was present in the start

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36
Q

what are the DNA amplification techniques

A
  • PCR
    -RT-PCR
  • qPCR
37
Q

what can qPCR do

A

quantify gene expression
- mRNA transcript of specific gene->cDNA
= # copies = expression of gene
- primer will only bind to gene of intrest and amplify that and outshine the og DNA
- though OG will still be present

38
Q

CT relationship

A

lower CT = more gene of intrest at start
low Ct= more expression of gene

39
Q

what are melt curves

A

validate specificity of qPCR reaction
- becuse qPCr should only be creating the gene of intrest

  • determine if sample is clean
    if all product melts at oncde them it is clean
  • would know the GC content so we know the Tm
40
Q

How is melt curve observed

A

each increase in temp should decrease the flouresences since SYBR wiill disscosiate when cDNA-> ssDNA

41
Q

molecular clonging steps

A
  1. plasmid generation
  2. recombinat vestor insertion
  3. positive and negative selection
42
Q

types of clonign vectors and their length of DNA theybcan take

A

Plasmid - 15 000 bp
BAC- 100 000- 300 000 bp

42
Q

molecular clonging what is positive and negative selection

A

postive selection- using antibiotic resistance, dircectly targeting and isolating product - targets gene, did the cell take in plasmid
- cells that have gained a specific gene survive

negative selection- removes unwated products leaving target product
- does not target gene,
- cells that have lost a specific gene survive

42
Q

DNA sequence anylysis techniques

A
  1. Snager sequencing
  2. dye terminator sequencing
  3. illumina- next gen sequencing
42
Q

SS- what are ddNTPS

A

chain elongation terminators- lack a 3’OH so DNA poly can not exztend

43
Q

concentration of dntp and ddntp for snager

A

dntp> ddntp
so that elongation can happen more then termination

43
Q

differnce btw sanger and dye terminator sanger and its advantages

A

DTS
- have flornce on ddntp
- ddntps all added at once (SS has 4 differnt reactions each with a ddNTP)
- use cappiliary gel electrophorisis (SS use gel electrohprisis to separte by sixe)

Advantages
- high throuput
- no radioactivity
greater read length

43
Q

what do doubke peaks on capillary electrophorissis mean

A

heterozygous or mutation becuse two pairs are read at same time

43
Q

Primer Design - known and unknown

A

for know sequnce - forward and reverse primers to known sequence

for unknow seq- ligateadapters then use primers that attcah to adapters

44
Q

next generation sequencing vs dye terminator
1. libary prep
2. amp
3. seq
4. ana

A
  1. DT- ligate fragments
    NG- ligate adpaters
  2. Dt- replicate plasmid containing bacteria
    ng- cluster geneeration
  3. dt- use ddntp
    ng- use reversible terminator ddntp
45
Q

NGS- what is the adpter have?
what is conserved ?

A

index read- barcode allowing for multiplexing

primer binding protein- for primer

terminal sequence - stick to snager

primer binding and terminal sequenc is conserved

46
Q

reading the clusterr generation
- coverage vs depth

A
  • coverage- is the amount of dna alinging want 100%
    depth is the amount of overlap
  • more over lap = more ecpression there for can indicate cancer or over ecpression
47
Q

What is RNA- seq

A

assement of alternitive splice barients

  • gene expression by counting molecules more depth = more read
48
Q

RNA-seq process

A
  • mrna->cdna
    -adpaters to cdna
  • align to genome only to exons
49
Q

genome organization

A

chormosome - telomer+ centermere
- p arm (petitie) and Q arm

chromatin- dna and protein

nucleosome - dna and protein ()histone

histone- 1H1: 2H2A: 2H2B: 2H3: 2H4
h2a/b are dimers
h3/h4 are tetramers
- basic to attach dna backbone

50
Q

pertsonlized medicine

A

when comparing the genome of a the same species to fine gene specific disease

51
Q

what is comparitive genomic

A

comparing the genes of differnt oraganisms
to:
Assign gene function
Contribution of specific gene to disease
Evolutionary history (phylogenetic tree)
Genetic basis

52
Q

compartive genomic terms to assign gene function

A

homologs- same sequence similairty (eveolution)

othologs- same sequence and funcrtion in differnt species (common ansector)

paralogs- related genes in same species - gene duplication

53
Q

how can evolutionary history occur
(2)

A

SNPs- single nucleotide polmorphism
subsitution of one nt
- such as chimps and humans
- most common

genomic rearrangments
- inserstion, deletion, invresion, duplication

54
Q

hwo are outgroups created

A
  • snps
  • they can show what use to be similar
55
Q

diffence between prokar/eukar for genome

A

prokaryots mostly functional dna with small introns and large exons or one exon

eukaryotes mostly non functional dna and 33% are coding genes (include regulatory sequencs intron and exons)
- large intons and small exons, repative sequences

56
Q

importance of centermeres and telomers

A

centermere - segragation site and place for kinetic core assembly

teleomers- prevetn degragation
- can not use hTERT (is increase in embryo, cancer and stem cells )

57
Q

what contributes to compaction (30nm filment )

A
  1. 6-7% N-terminal tails of histome
  2. H1 histone
  3. histon fold motif
58
Q

what interacts with the minor groove of DNA

A
  1. histones
  2. histone fold motif
59
Q

what is conserved for histones

A

H3-H4

60
Q

Kornberg discovery

A

each chromatin unit is ~200 bp
h3-h4 is tetramer and h2a/h2b are dimers

61
Q

requirments of DNA compaction system

A
  1. dynamic - to allow for closing and transcribing whenever needed
  2. modifiable- globala nd local
  3. reponsive- crc and histone modif
62
Q

regulation of DNA compaction (2)

A

Chromatin remodeling complex and histone modifying encymes

63
Q

Chromatin remodeling complex

A

displacement - open a transcription site and close another
eject- open a transcription site
replacement- chnage effecinty

64
Q

Replacment- Chromatin remodieling complex

A

H3 varients-
H3.3 - keep open state
cenpa- attach kinetic core

H2A varients
H2AX- dna damage repair by being able to be phosphoralated by ser139 which attracts dna repair proteins
macro H2A- long c trminal end that siliences one X gene

65
Q

histone fold motif

A

thre helicase linked by two short loops
10 bp segments = 1 full turn
interact with at of the minor groove
- non specific -

66
Q

n-terminal tails

A

flexable
form interactions with adjacent nucleosomes
less interactions with other nucleosomes increase dna acces and thre for transcription

67
Q

H1 linker histone

A

protects the 21 bp and attached the end and middle of the linker DNA

68
Q

most importance compaction to the 30nm filment

A

n-terminal tails

69
Q

glemsa stain

A

determine what is active and not
dark bands are heterochromatin= close and compact
eurochromatin= opena not compact beijgntranscriped

70
Q

caf-1 deliver H3-H4 during replication

nap-1 deliver h2a/h2b dring replication

A
71
Q

What happens if genome is ac/met during replication

A

the propeties willl somtimes belost,
- bromo or chromodomanins can proprogate those cheimical signals
- to all the rest of the hostones

72
Q

epigentics

A

the study of heritable traits
- do not incvolve chnages in dna but what is being expressed

73
Q

examples of epigentic modifications

A

materal / nutruring influence
chromatin remodeling
histone modifi
mitochondia

74
Q

Histone modifs

A

HATs- acetylate
HDACs- deacetlate

HMTs- methylate
Jumangi- demethylate

75
Q

acetlyation of lys

A
  • neutrilize charge
  • open the histone to transcription by weakening tail interactions
76
Q

methlyate arg anf lys

A
  • does not chnage charge
  • can be one two three methaylated
  • stablize open or closed state by binding preotein complexes
77
Q

ChIP assay

A

test if gene is active for transcription
- first by crosslinking dna and protein
- then put antibodys that bind the protein/ actyl / methyl

  • then use beads to separte protein with antibody an other bound proteins
  • separte dna from proteina ndimmunopercipatate the histones
78
Q

important activation and repression groups

A

activation- H3K9ac, H3K4me3
repression- H3K9me3

79
Q

Phosphoralation of ser, the,tyr

A
  • initaite recuirtment and relase of histone modifying enxymes or chromatin
80
Q

histone modifications cis vs trans

A

cis is direct tighting or lossoning of nucleosome

trans is indirect and use of preotien

81
Q

example of cis histone modifications

A

lysine acetylation

82
Q

example of trans histone modification

A

chromo or bromdomains

83
Q

Bromodomains

A

recognize HATs on lys

propergate acetyllation (open state)

84
Q

Chromodomains

A

recognize HMTs on lys

activation or repression of genes

85
Q

what is chip-Seq

A
  • next gen sequencing
    read will only align over segments bound to histones
    less read depth in areas with linker dna

BCHM 218 (2 decks)

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