IAS07

Question 1

human genome & chromosomes

Answer 1

all genetic info in body (20k protein coding)
3 billion base pairs, 2m long packed into nucleus
46 chromosomes: 22 pairs of autosomes, 2 allosomes

Question 2

genome packaging, cytoband & chromosome structure

Answer 2

in chromosomes, p (short) & q (long) arm
telomere: near end of chromosome
centromere: constricted region between p & q arms, links sister chromatids together
T&C comprise highly repetitive sequence for DNA replication
cytoband: segments of genome for easy reference, contains multiple genes

Question 3

gene structure

Answer 3

functional units of genome
contain exons & introns, regulatory sequence that determine gene expression

Question 4

functional genome properties

Answer 4

organised, properly regulated / expressed / stored, stable, copied accurately to next gen of cells

Question 5

diploid v haploid

Answer 5

diploid: 2 sets of chromosomes, 1 from mother, 1 from father
haploid: cells w/ 1 set of chromosomes
diploid: somatic cells
haploid: gametes e.g. sperm, oocyte

Question 6

cell division & types

Answer 6

make copies of genome
meiosis: make haploid gametes from diploid cells in germline
mitosis: make diploid copies of chromosomes

Question 7

genetic variation: significance & types

Answer 7

described DNA diff. btn people / cells
accounts for most observational phenotypes e.g. most diseases & health conditions
affect gene expression
copy no., structural, seq. level

Question 8

copy no. var.

Answer 8

gain or loss of chromosomes / whole arms
e.g. down syndrome, trisomy 21, 3 chr. 21
symptoms: distinctive facial features, delayed growth, mild-moderate mental retardation

Question 9

structural var.

Answer 9

gain or loss of large parts of chromosomes (1kB-3mB):
deletion, duplication, invertion, translocation (frag. spliced into another genome region)
e.g. facioscapulohumeral muscular dystrophy (FHMD): loss of chr. 4q35 in 30-3000kB, reduced D4Z4 genes, DUX4 expression & activation -> toxicity to muscle
adolescent onset, progressive muscle weakness of head, shoulder, arm

Question 10

sequence level var.

Answer 10

single nucleotide polymorphism / indels of small DNA fragments e.g. SCD

Question 11

DNA sequencing

Answer 11

goal: work out order of nt base i.e. determine arrangement of bases

Question 12

DNA replication (PCR)

Answer 12

in lab: DNAP adds base after base by CBP to create ds dna; specifically adds phosphodiester bond of 5’ of incoming nt & 3’ of nt of growing chain, with 2 Pi released -> elongation

Question 13

sanger sequencing materials

Answer 13

DNA nts mixed w/ fluorescently labelled dideoxy nts w/o -OH in 2’ & 3’, each dideoxy nt has different color
cannot be elongated hence since no phosphodiester bond formed in 3’ OH i.e. chain-elongation inhibitors of DNAP

Question 14

sanger sequencing steps

Answer 14

• mix DNA seq., DNA nts, small amount of dideoxy nts for primer elongation in PCR
• dideoxy nts randomly incorporate into growing chain -> chain termination -> DNA fragments of diff. sizes produced due to many PCR reactions occurring in parallel
• separation by size by gel electrophoresis
• identity of nucleotide identified in chromatogram

Question 15

limitations of sanger sequencing

Answer 15

works reliably at 500-800bp, else too labour-intensive & low throughput

Question 16

ng sequencing techniques

Answer 16

short reads: broken down into shorter fragments for seq., 150-300 bp by amplification w/ PCR e.g. sequencing by synth., nanoball sequencing, pH sensing
differ by DNA library preparation, sequencing chem & detection methods
long reads: single molecule real time sequencing SMRS, e.g. dye labels & nanopore sensing

Question 17

ng sequencing techniques advantages, disadvantages & current usage

Answer 17

short reads used due to low error rate, high thruput, cost advantage
limitation: short read size, need for PCR
short read usage: detect SNP in disease
long reads usage: detect chemical modification of DNA / large scale genome rearrangement, sequence RNA transcripts of few kB, assemble whole genome of novel organisms

Question 18

sequencing by synthesis: sample preparation

Answer 18

prepare sequencing libs, add adaptors & barcodes to DNA sequence
adaptors aid attachment of fragments to sequencing flow cell & amplification
barcodes for pooling of multiple samples into single expt for identification

Question 19

sequencing by synthesis: process

Answer 19

DNA seqs amplified on solid surface of flow cell -> fluorescently tagged DNA nts with 3’ blocked add to growing nt by CBP one at a time -> excited by light sources, base-specific fluorescent signal emitted = color of added nt
fluorophores cleaved off -> 3’ -OH group regenerated -> cycle repeats again
allows billions of nts synthesized in a massively parallel process

Question 20

cost of whole genome sequencing

Answer 20

fallen drastically to <1000 USD

Question 21

detect genetic variation with short read sequencing

Answer 21

sequencing read in fastq format -> align to reference genome -> calling of seq variants, determine diff btn seq. gene & reference gene