human genome project

Question 1

requirements for DNA

Answer 1

must carry information
must replicate
must allow for info to change
must govern the expression of the phenotype
extreme accuracy of DNA replciation is necessary to preserve the genome over generations
complementarity allows the heredity info to be copied digitally, accurately, quickly and efficiently + ensure mutation rates are low

Question 2

HGP

Answer 2

goal was identify the order of all the bases on every chromosome
develop new technology + resources, understand ethical, legal and social ussues
in 2003, HGP produced a genome sequence that accounted for over 90% of human genome
over 99.6% is done now

Question 3

HGP process

Answer 3

DNA from 5 human subjects used
genomic fragmentation to DNA fragments using restriction enzymes
genome fragments placed in vectors (BAC or plasmids)
first generation sequencing
supercomputer put the pieces together; assembling each BAC then entire sequence
contigs to scaffolds + scaffolds placed on BACs, scaffolds placed on chromosomes to generate whole genome assembly

Question 4

types of sequencing

Answer 4

hierarchical shotgun
whole genome shotgun

Question 5

hierarchical shotgun

Answer 5

lots of large insert clones and keeps them separate
genome is broken down into larger segments

Question 6

whole genome shotgun

Answer 6

whole genome is sequenced
sequences overlapping DNA sequences
computer is used to assemble the dna fragments

Question 7

genome assemblies hierarchical

Answer 7

genome assembly- the process of putting nucleotide sequence into the correct order.
shortest assembly components are contigs which are sequences taken from individuals
contigs are assembled into longer scaffolds + scaffolds are assembles into chromosome if there is sufficient mapping info

Question 8

types of scaffold

Answer 8

placed- placed within a chromosome
unplaced scaffold- not known which chromosome scaffold belongs to
unlocalised scaffolds- scaffolds orientation is not known

Question 9

gene size

Answer 9

genes vary in size and exon content
inverse correlation between gene size and fraction of coding DNA
8.8 exons + 7.8 exons per gene
natural selection favours short introns in highly expressed genes as transcription is costly in time and energy

Question 10

reference genome

Answer 10

digital nucleic acid sequence database, representative example of set of genes in one idealised individual organism
assembles from sequencing of DNA from a number of individual donors
improvements driven by technological advances

Question 11

human pangenome

Answer 11

contains 47 phased, diploid individuals from a cohort of genetically diverse indivduals (trying to sequence regions with lots of variation)
added 119 million base pairs to the exisitng reference GRCh389
set of reference human genome sequences

Question 12

Y chromosome

Answer 12

complete sequencing of Y chromosome
uncovered important genetic features including factors in sperm production
loss of Y chromosome is observed in multiple cancer types so can understand why
Y chromosome is highly repetitive

Question 13

acrocentric chromosomes

Answer 13

recombination
short arms of human acrocentric chromosomes (12,14,15,21 +22) share large homologous regions including ribosomal DNA repeats and extended segmental duplications
short arms become more like eachother as they swap DNA
mediated via the PHRs because they contain genes for making ribosomal RNAs

Question 14

robertsonian translocations

Answer 14

impact of lost segments of p arms
often seen in cancers
potential intranuclear mislocalisation of the q arms of the chromosomes

Question 15

genome engineering- pluripotent stem cells

Answer 15

1. isolate and culture donor cells
2. transduce (convert) stem cell-associated genes into the cells by viral vectors
3. harvest and culture the cells according to ES cell culture
4. a small subset of the transfected cells become iPS cells and generate ES like colonies

Question 16

parkinsons disease

Answer 16

neurodegeneratove pathology affecting dopaminergic pathway in CNS
PD is histologically marked by degeneration of midbrain dopamine neurones in substantia nigra
since one of the triggers of PD is the deterioration of dopaminergic neurons and the accumulation of Lewy bodies in the SN (substantia nigra), the maintenance of this cell population has become a therapeutic target.
neurotrophic factors have been proposed as a tool to enhance the neuroportection of this population

Question 17

genome enginerring- pluripotent

Answer 17

skin cells from tails of lab mice generate induce pluripotent stem cells (iPSCs)
can convert XX cells into primodial germ cells and take germ cells to generate egg cells in tissue culture
Some iPSCs in tissue culture spontaneously lose the Y chromosome, generating “XO” cells
XO cells can develop two X chromosomes because of cell division errors
Convert the XX cells into primordial germ cells
Take primordial germ cells and generate egg cells in tissue culture

Question 18

genome testing

Answer 18

BRCA1 and BRCA2 often mutated in breast cancer
lots of genome testing techniques that cna detect mutation in BRCA
genomic testing for newborn babies in ITU to identify rare genetic disease rapidly via whole gencome sequences (if babies have failure to thrive)

Question 19

gene editing- B globin locus

Answer 19

alpha and beta loci determine structure of 2 types of polypeptide chains in adult hbA
mutatnt beta globin causes sickle cell anaemia
sickle hb polymerises under hypoxic conditions where deformed red cells hemolyse and cause vasoclusion
genome editing of patient HSCs by clustered regularly interspaced short palindromic repeats (on target)

Question 20

CRISPr cas 9-gene editing on target

Answer 20

clustered regularly interspaced short palindromic repeats
sickle cell caused by single point mutation in which glutamine acid replaces valine in codon 6 of human beta globin gene
genome editing of CRISPr- disrupt cis regulatory elements by NHEj or correct HBB (human beta globin gene) by HDR(omology-directed repair)
GPH101 is a hemototpoeitic stem cell (HSC) drug product designed to correct the SCD mutation in the HBB gene using high fidelity cas9

Question 21

CRISPr cas 9

Answer 21

off target
remove repression of y- globulin via BCL11A
elevated foetal hb levels in red cells protect against complications of sickle cell disease
base and prime editords to disrupt cis regulatory elements or off set the mutation in HBB
OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9–edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product
targeted disruption of HBG1 and HGB2 gene promoters that increase foetal hb edxpression in red cell progeny

Question 22

gene therapy- alcohol use disorder

Answer 22

glial-derived neurotrphic factor (GDNF) into the region of the brain where dopamine exists
efficacy of GDNF inserted in reversing alcohol induced hypodopaminergia in the mesombolic system and suppressing ethenol intake
compared to the control group, consumption in monkeys dropped by 90%
gene effectively reset monkeys brain reward pathways

Question 23

ethical legal and social issues

Answer 23

privacy and confidentiality of genetic info
fairness in the use of genetic info by insurers, employers, courts etc
psychological implicaition, stigmatization and discrimination due to genetic differences
reproductive issues including adequate and informed consent and use of genetic info in reproductive decision making

Question 24

gene therapy- car T

Answer 24

• Ex vivo gene therapy refers to the genetic manipulation of cells outside the body
• In cancer, it involves removing cells from a patient, culturing and modifying them in a laboratory to give them the ability to destroy cancer cells, and delivering them back to the patient
• Chimeric Antigen Receptor T (CAR-T) cells are the most commonly used cells
• T-cells from the patient are modified using a virus that codes for a gene that produces a receptor, called Chimeric Antigen Receptor (CAR), hence the name CAR-T cells
• This receptor varies for each cancer type, is specific to cancer cells, and gives T cells the ability to specifically bind to cancer cells without harming healthy cells