genetic variation and detection

Question 1

why we care ab genomic variation

Answer 1

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

Question 2

benefits- molecular medicine

Answer 2

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

Question 3

benefits - bioarcheology, evolution + human migration

Answer 3

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

Question 4

benefits- dna identification

Answer 4

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

Question 5

benefits - agriculture, livestock breeding + bioprocessing

Answer 5

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

Question 6

benefits - microbial genomics

Answer 6

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

Question 7

main types of variation

Answer 7

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

Question 8

numerical abberation

Answer 8

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

Question 8

structural aberration

Answer 8

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

Question 8

tandem repeat polymorphisms

Answer 8

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

Question 9

insertion/deletion polymorphisms

Answer 9

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

Question 10

single nucleotide mutation

Answer 10

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

Question 11

dna structure

Answer 11

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

Question 11

human genome organisation

Answer 11

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

Question 12

genome comparison

Answer 12

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

Question 13

repetitive sequences

Answer 13

unique or non repetitive sequences
moderately repetitive
highly repetitive

Question 14

unique or non repetitive sequences

Answer 14

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

Question 15

moderately repetitive

Answer 15

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

Question 16

highly repetitive

Answer 16

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

Question 17

dna polymorphisms

Answer 17

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

Question 18

common dna polymprophisms

Answer 18

tandem repeats
interspersed repeats
length polymorphisms

Question 19

tandem repeats

Answer 19

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

Question 19

interspersed repeats

Answer 19

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

Question 20

what markers should we study

Answer 20

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

Question 20

length polymorphisms

Answer 20

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

Question 21

biochemical markers

Answer 21

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

Question 22

molecular/dna genetic markers

Answer 22

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

Question 23

dna extraction

Answer 23

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

Question 24

dna requirements- quantitiy

Answer 24

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

Question 25

dna extraction process

Answer 25

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

Question 26

cellular lysis

Answer 26

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

Question 27

which method should be used

Answer 27

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

Question 27

dna isolation

Answer 27

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

Question 27

lystate cleaning

Answer 27

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

Question 27

common extraction methods

Answer 27

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

Question 28

organic method

Answer 28

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

Question 29

salting out

Answer 29

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

Question 30

solid phase silica/ spin column

Answer 30

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

Question 31

chelex method

Answer 31

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

Question 32

FTA cards/ FTA elute

Answer 32

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

Question 33

dna quantification

Answer 33

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

Question 34

goals of quantification

Answer 34

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

Question 35

spectrophotometry

Answer 35

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

Question 36

spectrophotometry adv and dis

Answer 36

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

Question 37

fluorescent dyes

Answer 37

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

Question 38

fluorometric adv and dis

Answer 38

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

Question 39

electropheresis

Answer 39

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

Question 40

electropheresis analysis

Answer 40

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)

Question 41

electropheresis adv and dis

Answer 41

adv - specific sized bands - impure samples dis - need lots of dna - not highly accurate

Question 42

polymporphism detection methodolgy

Answer 42

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

Question 43

PCR amplification

Answer 43

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

Question 44

PCR components

Answer 44

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

Question 45

dna amplification

Answer 45

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

Question 46

pcr thermocyclers

Answer 46

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

Question 47

factors impacting PCR

Answer 47

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+

Question 48

convention pcr- alu analysis

Answer 48

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