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Genomics > genetic variation and detection > Flashcards

genetic variation and detection Flashcards

1
Q

why we care ab genomic variation

A

genetic variations underlie phenotypic differences among different individuals
determine our predisposition to complex diseases and responses to drugs + environmental factors
reveals clues of ancestral human histiry

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2
Q

benefits- molecular medicine

A

improve diagnosis of disease detect genetic predispositions to disease
create drugs based on molecular info

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3
Q

benefits - bioarcheology, evolution + human migration

A

study evolution through mutations in lineages
study migration of different populations based on maternal inheritence
study mutations on Y chromosome to trace lineage and migration of males
compare breakpoints in the evolution of mutations with ages of populations and historical events

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4
Q

benefits- dna identification

A

identify potential suspects whose DNA matches evidence
identify endangered species
establish paternity and other family relationships

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5
Q

benefits - agriculture, livestock breeding + bioprocessing

A

grow disease, insect and drought resistant crops
breed healthier, more productive disease resistant farm animals
grow more nutritious produce

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6
Q

benefits - microbial genomics

A

rapidly detect and treat pathogens
develop new energy sources
monitor environments to detect pollutants

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7
Q

main types of variation

A

gross chromosomal abberation (macro)- numeical + structural aberration
insertion/ deletion of >1 nucleotide (medium)- tandem repeat polymorphism, insertion/deletion polymorphisms
single nucleotide mutation (micro)

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8
Q

numerical abberation

A

usually cuased by a failure of chromosome division, results in cells with an extra chromosome or deficiency in chromosomes
gametes with these anomolies can result in down or turner syndrome
include- triploidy 2 sets, tirsomy; 3 copies of 1 chromosome, monosomy; 1 copy of chromosome or masochism; different sets of cells in the body

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8
Q

structural aberration

A

deletioms, inversions or translocations of large DNA fragments
both types of macro mutation are rare but often causing serious genetic disease

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8
Q

tandem repeat polymorphisms

A

genomic regions consisting of variable length of sequence motifs repeating in tandem with variable copy number
used as genetic markers for DNA profiling
microsatellites- short tandem repeats- repeat unit 1-5 bases long
minisattelites- repeat unti 11-100 bases long

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9
Q

insertion/deletion polymorphisms

A

indel or dips
often resulted from localised rearrangements between homologous tandem repeats

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10
Q

single nucleotide mutation

A

resulting in a single nucleotide polymorphism/ variant
accounts for up to 90% of human genetic variations
majority of SNPs do not directly or significantly contribute to any phenotypes
each SNP has a specific location on the genome referred by RS number

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11
Q

dna structure

A

two polynucleotide strands wound around each other
backbone consists of alteration deoxyribose and phosphate groups
dna strands assembled in 5’ to 3’ direction and by convention are read the same way
purine and pyrimidine attached to each deoxyribose projects in toward the axis of the helix
each base forms h bonds with opposite= base pairs
double helix makes a complete turn in just over 10 nucleotide pairs, providing stability
path taken by backbone forms a major groove and minor groove

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11
Q

human genome organisation

A

genome is made up of coding and non coding regions
coding= genes and proteins or RNA
non coding= introns, regulatorrs + repetitive dna
repetitive dna can be intersperesed or tandem

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12
Q

genome comparison

A

eukaryotic genomes vary substanitally in size
in many cases, variaion is not related to the complexity of the species but rather the accumaltion of repetitive dna sequences (commonly non coding)
3 main types of repeititve sequences- unique or non repetitive, moderately repetitive or highly repetitive

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13
Q

repetitive sequences

A

unique or non repetitive sequences
moderately repetitive
highly repetitive

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14
Q

unique or non repetitive sequences

A

found once or a few times in the genome
includes structural genes as well as intergenic areas

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15
Q

moderately repetitive

A

found a few hundred to thousna times
genes for rRNA or histones
origins of replications
transpisable elements- mobile dna sequences capable of replication themselves with genomes independently of host cell

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16
Q

highly repetitive

A

found 10s of thousands to millions of times
each copy is relatively short
some sequences are interspersed throughout the genome
other sequences are clustered together in tandem arrays

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17
Q

dna polymorphisms

A

are the different dna sequences among individuals, groups or populations- polymorphisms at the dna level include a qide range of variatio ns
sequence compared to a reference standard that is present in at least 1-2% of the population

can be single base or thousands of bases
if the location of a polymorphism is found, can be a landmark for locating other genes
each polymorphic marker has different versions or alleles

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18
Q

common dna polymprophisms

A

tandem repeats
interspersed repeats
length polymorphisms

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19
Q

tandem repeats

A

copies which lie adjacent to each other, either directly or inverted
satellite dna- typically found in centromeres and heterochromatin
ministallite- repeat units from about 10 to 60 base pairs- found in many places in the genome including centromeres
microstallite- repeat units of less than 10 base pairds include telomeres which typically hav e 6 to 8 base pair repair units

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19
Q

interspersed repeats

A

dispersed throughout the genome
aka interspersed nuclear elements
transposable elements
retrotransposoms
LTR retro transposons
non LTR retrotransposons - SINES + LINEs short and long interspersed nuclear elements

20
Q

what markers should we study

A

patterns of inheritence- well established
independent inheritence
polymorphic and heterozygous
simple, rapid and reproducible detection
small amount of material needed

20
Q

length polymorphisms

A

RFLPs- restriction fragment length polymorphisms - original method of SNP analysis
VNTRs- variable number tandem repeats- large size polymorphisms, require high quality
STRs- short tandem repeats
Alu- transposable elements
copy number variants

21
Q

biochemical markers

A

blood groups, HLA, serum proteins + red cell enzyme; low polymorphism but still have utility

22
Q

molecular/dna genetic markers

A

currently very popular, high polymorphism + finest level of variation, relatively easy to analyse- can identify down to single individual

23
Q

dna extraction

A

to obtsin dna in a relatively purified form which can be used for further investigation
dna sources: included any nucleated cell, body fluids (blood, saliva, sweat) tissue (hair roots, fingerprints)
amount of dna varies from source to source- blood= 30mg/ml hair= 70ng/ml

24
Q

dna requirements- quantitiy

A

diploid cell contains approx 6pg of dna
average adult has 5x10^6 wbc per ml of blood
recovery of dna per ul of blood is 30-60ng
pcr reactions require 1-50ng of dna on average

25
Q

dna extraction process

A

dna is well protected so it needs to be processed before we can analyse it
different methodologies for extracting dna from bniological samples but most often they can be divided into 3 phases
1. cellular lysis via disruption of cell membranes
2. protein removal- lysate clearing
3. dna isolation from the remaining solution

26
Q

cellular lysis

A

breaking of cell membrames- plasma and nuclear
detergents can break down cell membrane
detergents disrupt membranes due to amphipathic (hyrophobic + hydrophillic) nature of both cellular membrane and detergent molecules
detergents contains SDS
result of the lysis is that cellular contents are distributed in solution

27
Q

which method should be used

A

for dna isolatiom
selection of the most suitable method for sample preparation depends on the type of sample and purpose of the molecular analysis
no universal approach
consider
- sample matrix/quality
- prep method
- intended use
- sample quantity
- yield
cost, time, simplicicity, dna quality + safety

27
Q

dna isolation

A

involves addition of salts
salts interupt the h bonds between water and dna molecules
dna is precipitated using isoproponal or ethanol
dna is commonly pelleted by spinning with a centrifuge and the supernatent is removed

27
Q

lystate cleaning

A

removes proteinasceous material
commonly done by denaturation and precipitation of proteins
as we also precipitate dna from our cellular supernatent we can term the process differential denaturation or differential precipitation
can use phenol/ chloroform or specific salts and buffers to remove proteins from dna

27
Q

common extraction methods

A

organic- best quality of dna but laborious
salting out- good quality and less use of hazardous chemicals
solid phase silica extraction- dna spin column technology enables nucleic acid purification by using solid phase silica extraction
ionic chelating resins eg chelex- easy short and quick method- used for pcr
fta method

28
Q

organic method

A

place sample in tube
add SDS and proteinase K to rupture cells
incubate
add phenol chloroform
vortex and centrifuge
dilute in water- transfer and retain aqueous phase dna

29
Q

salting out

A

isolate nuclei pallets (lysis buffer + centrifugation)
overnight proteinase K digestion
saturated (chaotrophic) salt solution to salt out digested protein (dna highly hydorphillic due to phosphate backbone)
retains supernatent and discard pellet
dna precipitation by mixing supernatent in ice cold ethanol
dilute in water or TE and store for further analysis
high salt= possible comtamination

30
Q

solid phase silica/ spin column

A

collect epithelial cells from buccal activity
lyse- disruption of cells by chaotropic salts, detergens or alkaline denaturation
bind-dna contained within the lysate will bind to the silica column in the presence of chaotropic salts
wash- post binding the resulting lysate is cleared by centrifugation
elute- removal of salt solution to enable DNA to be released by the silica bed
results in ready to be purified dna

31
Q

chelex method

A

chelating resin (styrene divinylbenzene copolymers with paired iminodiacetate ions)
only suitable for pcr so has limited end application processes

32
Q

FTA cards/ FTA elute

A

chemical coatings of FTA cards
dna can remain bound to the vard or be eluted using water and heat
direct amplification (no washing)
standard amplification (with washing)
extraction or purification of DNA from card
expensive and proprietary

when cells are applied to FTA cards the cells are lysed and the nucleic acids are immobilised and stabilised within the cards matrix

33
Q

dna quantification

A

assess quality of DNA
determine amounts needed for downstream applications - sequences, PCR and cloning
confirm experiement/ extraction

34
Q

goals of quantification

A

concentrate the target organisms/ sample and subsequently the template dna
maximise the quality of dna to produce homogenous sample to enable specific and sensitive analyses

35
Q

spectrophotometry

A

spectrophotometer equipped with UV lamp
biomolecules absorb light in UV range (dna 260-280nm, proteins 215-230 and 280nm)
analysis of UV absorption by nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample
purines and pyrimidines in nucleic acid show absorption maxima around 260nm
absorbance in UV range allows us to estimate amoint fo DNA by its absorbance
common UV spec is nanodrop

36
Q

spectrophotometry adv and dis

A

adv
- used small microvolumes (1-2ul)
- rapid results for quick assessments
- widely used

dis
- not species specific
- bad resolutionnfor low conc samples
- doesnt distinguish between dsdna and ssdna
- contaminating samples lead to falsely high quantification readings

37
Q

fluorescent dyes

A

dna dyes are fluorescent dyes that bind nucleic acids- normally bind to dsDNA
many applications including flow cytometry, cell cycle studies, dna sequencing + quantifying
once dye is mixed and incubated with dna sample you can measure fluorescent intensity of dye with a spectrophotometer
dna quantified by comparing sample to set of standards
qubit- useds qubit fluorescence dye to determine concentration, higher fluorescent signal = higher dna quality
classes of nucleic acid stains- intercalating dyes, minor groove binders

38
Q

fluorometric adv and dis

A

adv
- high thoroughput
- increased sensitivity
- enhanced specificity compared to UV spec
- popular as it is simpler and more sensitive

dis
- need special equipment + reagents
- longer prep time than straight UV absorbance

39
Q

electropheresis

A

aragose gel often use for size fractionation
movement of charged particels in fluid or gel under the influence of an electric field- separating molecules in medium
differentiation on thr basis of size + nature of dna
ethidium bromide staining can then be carried out in DNA embedded within an aragose gel
visualisation of DNA via fluorescence using specific dyes - fluorescent yield of dye:dna complex is greater than unbound dye
more dna = more absorbance

40
Q

electropheresis analysis

A

calculate band sizd using software from transillimuniator
compare fluorescent intensities of ladder and sample to estimate dna concentration
create graph with linear trendline to calculate mass v intensity (standard curve)

41
Q

electropheresis adv and dis

A

adv
- specific sized bands
- impure samples

dis
- need lots of dna
- not highly accurate

42
Q

polymporphism detection methodolgy

A

PCR
most powerful tool in molecular biology
advances due to PCR- genomic sequencing, forensic dna profiling, disease diagnosis

43
Q

PCR amplification

A

PCR is a quick way to amplify a small segment of DNA
its operation relies on a DNA poly called taq (polly)
Taq is a polymerase made from thermophilus aquatiscum (bacterium)
PCR primers- normally 2- are required to dictate the segment of DNA amplified

44
Q

PCR components

A

primers- short strands of DNA which are complementary to the target sequence
nucleotides- single units of bases which are building blocks for new DNA strands
taq poly- thermostable enzyme that synthesises new strands of dna complementary to target sequence
buffer- contains mag ions + works as a cofactor for the dna pily and assits in the addition of nucleotides

45
Q

dna amplification

A

denaturation of dna strands- heating to 94-98c to break h bonds
annealing of primers to dna- lowered to 50-65c to allow annealing
extension by taq polymerase- 72c (optimum temp for taq)
steps are repeated between 32 -40 times in cyclical process

46
Q

pcr thermocyclers

A

allows us to change temp for each step
performs all steps in a cycle and repeats
denature (95C) , annel (60C), extend (72C)

47
Q

factors impacting PCR

A

no of cycles- more cycles + more dna (limited bny no. of nucleotides in a sample)
changing annealing temp- primers have optimal annealing temp, too low can lead to off target annealing and too high can lead to no primer binding
alter mg2+ conc- mg ions have a variery of effects and the fidelity of the pcr depends on mg2+

48
Q

convention pcr- alu analysis

A

analysis of transposable elements- detect oif one has been inserted or not
make up 6-13% of total dna with each one approx 300bp per copy
most prevelent is alu element
highly conserved, inserted in the last 10000 years
dimorphic, biallelic, dialleliec

Genomics (11 decks)

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