microarrays and dna sequencing

Question 1

dna library

Answer 1

set of overlapping dna clones
contains dna representing the entire genome of an organism
once library is created, it is screened for a particular gene of interest
screening is based on homology between a probe and one of the clones

Question 2

cloning

Answer 2

massive amplification of dna or rna sequences
stable propagation of dna sequences
single dna moleucle can be amplified allowing it to be:
studied- sequenced or hybridised
manipulated- mutagenised or engineered
expressed- generation of protein

Question 3

approaches

Answer 3

allows a copy of any specific part of dna or rna sequence to be selected among many others and produced in an unlimited amount
can be achieved by pcr- in vitro
or by cell based (traditional cloning) in vivo
both methods allow us to copy specific parts of dna or rna and produce an unlimited amount

Question 4

cell based cloning

Answer 4

5 steps
1. enzyme digestion of dna sample (RE)
2. enzyme digestion of dna plasmid vector
3. ligation of dna fragments and plasmid
4. transformation with ligation productions (into bacteria)
5. selection of prescence of products (growth on agar plate, blye white selection)
blue white- bacteria with inserted dna of interest will remain white

Question 5

microarrays

Answer 5

arrays/chips
orderly arrangement of thousands of spots or locations where minaturised chemical reactions take place, each spot contains 10^12M amount of dna
matching of known and unknown dna samples is done based on base pairing rules
each reactiom takes place at certain location to allow for identification
each of the locations is an immobilised target (probe) which is hybridised with by a complementary sequence
these can detect a range of analytes including dna, antibodies or protein

Question 6

dna microarrays

Answer 6

rely on hybridisation properties of nucleic acids to monitor dna or rna abundance on a genomic scale of different types of cells
relies on the complementary binding of dna (same as pcr)
probes are immobilised onto a solid surface eg glass
arrays can detect almost everything eg mrna, microrna, dna methylation etc
evolved from southern blotting but is a small scale equivalent

Question 7

dna microarray- sample prep

Answer 7

collection
- can be cell/tissue of organism we’re analysing
- can be different samples to be compared
(for gene expression need to isolate then convert rna to cdna via reverse transcription)

samples are amplified via pcr or cell based cloning
samples prepared for analysis- usually requires fluorescent dye- then samples added to microarray
target material hybridises to probe + excess material is washed away- laser scans microarray producing fluorescence, images are captured and converted to signal

Question 8

dna microarray image

Answer 8

meaning of symbols
P= pixel intensity
F=feature intensity
B= background intensity
Rp= ratio.of pixel intensities
Rm= ratio of means
mR= median of ratios
rR= regression ratio
image will show differential pattern of fluorphones so look at the different sports on the chip and try to determimne the relative intesnity of the pixels + create an image

Question 9

SNP array

Answer 9

single nucleotide polymorphism
identifying snps among alleles within or between populations
applications- genotyping, forensic analysis, identifying drug candidates

Question 10

array comparative genomic hybridisation (CGH)

Answer 10

assessing genome content in different cells or closely related organisms
prescence or absence of different material
testing a disease cell compared to healthy cell
- look for different colours produced
- eg green= pathological, red= healthy cell

Question 11

gene expression profiling

Answer 11

monitoring expression levels for thousands of genes simultaneously

Question 12

SNP microarray

Answer 12

target prep= amplify sample to create fragmemts
hybridisation= mix nucleic acids of target dna into array containing immobilised allele-specific oligonucleotides)
ligation-differentiate- labelled probes bind to sample, differentiating between the two alleles
signal amplification- make it bright enough + measure the intesnity

Question 13

microarray challenges/limitations

Answer 13

amound of dna in spot is not consistent
spot contamination
cDNA may not be proportional to that in the tissue
low hybridisation quality
measurement errors
outliers

Question 14

chromatin immunoprecipitation on chip

Answer 14

ChIP
dna seqeunces bound to a particular protein can be isolated by immunoprecipitatiing that protein
fragments hybribidsed to microarray
allows determination of protein binding site occupancy throughout genome

Question 15

dna methylation array

Answer 15

epigenetic modification in humans and many other species is dna methylation
methyl group is usually though to prevent transcription by recruiting protein complexes that either condense chromatin or act as a physical barrier against assembly of the transcription complex

Question 16

genomic sequencing

Answer 16

sequence of an individual’s genome is unique (expecte mono twins)
total of ~0.1% variation in the genome between individuals
human nuclear genome is over 3 billion nucleotides long
genome sequencing is figuring out the order of dna nucleotides in a genome

Question 17

sequencing technologies

Answer 17

3 categories
1st gen= sanger sequencing
2nd gen= next generation sequencing
3rd gen= emerging technologies (sequencing the whole genome rather than breaking it down and looking at it individually)
most techniques involve breaking the genome down into sequencable fragments
HGP used 1st gen

Question 18

1st gen sequencing

Answer 18

preparation of dna
- digest genome to produce short fragments (using REs)
- amplify fragments - in vivo cloning
- generates large numbers of single unique colonies

has paralleles to pcr as it involves template dna, a primer, dna poly and nucleotides (dNTP and ddNTP)

Question 19

1st gen process

Answer 19

dna chains are synthesises on a template strand
chain growth is stopped when 1 of 4 possible dideoxy nucleotides, which lack 3’ hydroxyl group, become incorporated, preventing the addition of another nucleotide
a population of truncated dna molecules is produced that represent each of the sites of that nucleotide in the template dna
truncated dna molecules are labelled with fluorescent tags + separated by size

Question 20

2nd gen sequencing

Answer 20

various names including next generation sequencing, massively parallel sequencing, deep sequencing
various manufacterers have commonalities and produce the same output but have different detection chemistries
eg illumina and thermo

Question 21

2nd gen process

Answer 21

clonal generation - shotgun sequencing of random fragmented genomic dna or cdna reverse trasncribed from rna is performed without the need for cloning via a foreign host cell- linker or adapter sewuences are ligated to fgDNA or cDNA for contruction of template libraries

clonal amplification- performed on solid surface while isolated within miniature emuslion drops or arrays

nucleotide sequencing- nucleotide incorportation is monitored directly by luminescence detection or by changes in electric change during sequence proceudre

sequence alignment and data analysis- generates many millions of nucleotides short reads in parallele in much shorter time than sanger

Question 22

3rd gen sequencing

Answer 22

advances have led to development which offer sequencing of single molecules
can read
- long reads with no amplification
- detection of epigenetic markers without the need to modify dna
- access to genomic regions preveiously difficult to analyse

two main systems
- pacific biosciences (pacbio), single molecule real time (smrt) sequencing
- oxford nanopore technologies

Question 22

pac bio smrt

Answer 22

two modes available
- continuous long read with reads of >100kb with low accuracy
- circular consensus sequencing with reduced read lengths but increased accuracy from multiple reads

Question 23

ONT sequencing

Answer 23

dsDNA is unwound by the motor protein and one strand is passed through the nanopore to the positive side of the membrane
this process causes a change to the ionic current that is base specific

Question 24

sequencing coverage

Answer 24

2 factors when performing sequencing are the depth and breadth of coverage
depth can be calculated= read length x number of reads/ genome length
breadth is amount of genome being covered at a certain depth- referred to as % of the bases the reference genomes that are covered at a depth

Question 25

sequencing applications

Answer 25

whole genome sequencing
whole exome sequencing- only sequence protein coding genes, allows idenitification of genetic variants which are responsible for altering portein sequences/ structure in medelian and polygenetic disease
target region sequencing- sequencing of subset of genes or regions involved in a disease/ phenotype
various applications for dna sequencing includning epigenetics