Module 3 Flashcards
(93 cards)
1
Q
nucleoside vs nucleotide
A
Nucleoside- nitrogenous base + pentose sugar (adenosine, guanosine, cytidine, thymidine, uridine)
Nucleotide- phosphates + pentose sugar + nitrogenous base
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
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
4
Q
how were the dimensions and water content of DNA determined
A
X-ray crystallography by Rosaland franklin
5
Q
what is one helical turn of DNA
A
10 bp and 34A
6
Q
main charteristic of RNA
A
transient and extra hydrogen for H-bonds
7
Q
coding RNA =
A
1.5%
8
Q
Non-coding RNA =
A
85%
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
10
Q
Hair pin
A
- most common
nt arranged to maximize H-bonding and base stacking
11
Q
internal loop
A
dsRNA separte due to lack of watson crick
12
Q
DNA stability
A
- hydrogen bonding
- hydrophobic stacking and vanderwaal forces
- Ionic interactions
13
Q
How does Hydrogen bonding effect DNA stability
A
hydrogenbonding keeps the two strands together
- A-T allows for more compressibility
14
Q
what disrupts H-bonding
A
organic solvents
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
16
Q
what forces cause DNA stacking
A
van der walls
17
Q
How does ionic interactions effect DNA stability
A
Na+ and Mg++ stabilizes the backbone of DNA
- neutrilize the electrostatic repulsion
18
Q
extincition coefficient units
A
g/cm/L
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
20
Q
hyperchromicity
A
the increase of absorbance
nucleotides -> ssdna -> dsdna
21
Q
increase in absorbance from ssdna to dsdna
A
40%
dsDNA x 1.4 = ssDNA absorbance
21
Q
what does ph do to dna
A
high ph causes deprotonation of nt = reduced h bond
increase ph = decrease stability
22
Q
DNA hybirdization depends on
A
stringency and can form btw DNA-RNA DNA-DNA and RNA-RNA
23
Q
What is stringency
A
how much of conditions of the experiment allow for imperfect pairing of molecules
24
low stringency =
allow for mismatch pairing becuse the conditions are favorable
increase salt
decrease temp
no os
25
high stringency=
to anneal 100% complementnary the conditions dont have to be favorable
decrase salt
increase temp
yes os
26
PCR- equelence of in vitro to vivo
vivo= primase, pol 3, helicase
dna primers, taq polymerase , heat
27
PCR steps
denaturation, reannealing, elongation, repeat
28
primer design features
- length, gc conten, Ta, GC residues on 3'
- 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
29
polymerase catalyze rate
1000 bp/min
30
equation for PCR product
2^x
x= cycles
31
How to visulize PCR
agrose gel electrophorisis
- denaturing solution
- separtion by size- small run faster
32
How to optimize PCR (stingency + why)
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
32
ethimine bromide
used to visulize in PCR
- intercalaytes between dna
33
what is RT-PCR
PCR but on RNA and will quantify mRNA levels
34
What is end point PCR
only anazlyse the end product do know know wjat is in each step
- is the dna templatte present
35
q-PCR
real time can quanitfy after each step
- use flouresents
- how much template was present in the start
36
what are the DNA amplification techniques
- PCR
-RT-PCR
- qPCR
37
what can qPCR do
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
CT relationship
lower CT = more gene of intrest at start
low Ct= more expression of gene
39
what are melt curves
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
How is melt curve observed
each increase in temp should decrease the flouresences since SYBR wiill disscosiate when cDNA-> ssDNA
41
molecular clonging steps
1. plasmid generation
2. recombinat vestor insertion
3. positive and negative selection
42
types of clonign vectors and their length of DNA theybcan take
Plasmid - 15 000 bp
BAC- 100 000- 300 000 bp
42
molecular clonging what is positive and negative selection
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
DNA sequence anylysis techniques
1. Snager sequencing
2. dye terminator sequencing
3. illumina- next gen sequencing
42
SS- what are ddNTPS
chain elongation terminators- lack a 3'OH so DNA poly can not exztend
43
concentration of dntp and ddntp for snager
dntp> ddntp
so that elongation can happen more then termination
43
differnce btw sanger and dye terminator sanger and its advantages
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
what do doubke peaks on capillary electrophorissis mean
heterozygous or mutation becuse two pairs are read at same time
43
Primer Design - known and unknown
for know sequnce - forward and reverse primers to known sequence
for unknow seq- ligateadapters then use primers that attcah to adapters
44
next generation sequencing vs dye terminator
1. libary prep
2. amp
3. seq
4. ana
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
NGS- what is the adpter have?
what is conserved ?
index read- barcode allowing for multiplexing
primer binding protein- for primer
terminal sequence - stick to snager
primer binding and terminal sequenc is conserved
46
reading the clusterr generation
- coverage vs depth
- 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
What is RNA- seq
assement of alternitive splice barients
- gene expression by counting molecules more depth = more read
48
RNA-seq process
- mrna->cdna
-adpaters to cdna
- align to genome only to exons
49
genome organization
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
pertsonlized medicine
when comparing the genome of a the same species to fine gene specific disease
51
what is comparitive genomic
comparing the genes of differnt oraganisms
to:
Assign gene function
Contribution of specific gene to disease
Evolutionary history (phylogenetic tree)
Genetic basis
52
compartive genomic terms to assign gene function
homologs- same sequence similairty (eveolution)
othologs- same sequence and funcrtion in differnt species (common ansector)
paralogs- related genes in same species - gene duplication
53
how can evolutionary history occur
(2)
SNPs- single nucleotide polmorphism
subsitution of one nt
- such as chimps and humans
- most common
genomic rearrangments
- inserstion, deletion, invresion, duplication
54
hwo are outgroups created
- snps
- they can show what use to be similar
55
diffence between prokar/eukar for genome
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
importance of centermeres and telomers
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
what contributes to compaction (30nm filment )
1. 6-7% N-terminal tails of histome
2. H1 histone
3. histon fold motif
58
what interacts with the minor groove of DNA
1. histones
2. histone fold motif
59
what is conserved for histones
H3-H4
60
Kornberg discovery
each chromatin unit is ~200 bp
h3-h4 is tetramer and h2a/h2b are dimers
61
requirments of DNA compaction system
1. dynamic - to allow for closing and transcribing whenever needed
2. modifiable- globala nd local
3. reponsive- crc and histone modif
62
regulation of DNA compaction (2)
Chromatin remodeling complex and histone modifying encymes
63
Chromatin remodeling complex
displacement - open a transcription site and close another
eject- open a transcription site
replacement- chnage effecinty
64
Replacment- Chromatin remodieling complex
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
histone fold motif
thre helicase linked by two short loops
10 bp segments = 1 full turn
interact with at of the minor groove
- non specific -
66
n-terminal tails
flexable
form interactions with adjacent nucleosomes
less interactions with other nucleosomes increase dna acces and thre for transcription
67
H1 linker histone
protects the 21 bp and attached the end and middle of the linker DNA
68
most importance compaction to the 30nm filment
n-terminal tails
69
glemsa stain
determine what is active and not
dark bands are heterochromatin= close and compact
eurochromatin= opena not compact beijgntranscriped
70
caf-1 deliver H3-H4 during replication
nap-1 deliver h2a/h2b dring replication
71
What happens if genome is ac/met during replication
the propeties willl somtimes belost,
- bromo or chromodomanins can proprogate those cheimical signals
- to all the rest of the hostones
72
epigentics
the study of heritable traits
- do not incvolve chnages in dna but what is being expressed
73
examples of epigentic modifications
materal / nutruring influence
chromatin remodeling
histone modifi
mitochondia
74
Histone modifs
HATs- acetylate
HDACs- deacetlate
HMTs- methylate
Jumangi- demethylate
75
acetlyation of lys
- neutrilize charge
- open the histone to transcription by weakening tail interactions
76
methlyate arg anf lys
- does not chnage charge
- can be one two three methaylated
- stablize open or closed state by binding preotein complexes
77
ChIP assay
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
important activation and repression groups
activation- H3K9ac, H3K4me3
repression- H3K9me3
79
Phosphoralation of ser, the,tyr
- initaite recuirtment and relase of histone modifying enxymes or chromatin
80
histone modifications cis vs trans
cis is direct tighting or lossoning of nucleosome
trans is indirect and use of preotien
81
example of cis histone modifications
lysine acetylation
82
example of trans histone modification
chromo or bromdomains
83
Bromodomains
recognize HATs on lys
propergate acetyllation (open state)
84
Chromodomains
recognize HMTs on lys
activation or repression of genes
85
what is chip-Seq
- next gen sequencing
read will only align over segments bound to histones
less read depth in areas with linker dna
