Chp 2 Lec1+2+3

Question 1

Sequencing is now done under a variety of auspices:

1. International HapMap project, focused on___ and collected__?
2. 1000-genome project, extended the HapMap project towards___ to focus on___. They sequenced over____.
3. Genomics England, goal is to sequence?
4. Personal genome project, goal is to? Likewise, US NIH supports?

Answer 1

Variations in genome sequences among populations distributed around world. And an atlas of SNPs and clusters of SNPs(I.e.haplotypes)

Complete genome data, conditions required to ensure appropriate data quality. 2500 individuals;family groups (mother + father + child) and 1000 protein-coding regions in 1000 individuals.

100000 genomes, including tumor and healthy cells from cancer patients.

Support developments in genome-informed and personalized medicine by publishing complete genomes and medical records of 100000 volunteers.
A project for 200000 human genomes

Question 2

Other cases of human genome sequencing:

1. Sequence determination of particular genes:
2. Personal genome sequencing @private companies:
3. Secondary school in New Jersey (USA):
4. Sushi:

Answer 2

1. • BRCA1, BRCA2 and PALB2, breast cancer predisposition
     
     • mutations correlate with early development of breast and ovarian cancer
     • renowned case, A Jolie- mutation in BRCA1; prophylactic double mastectomy, ovaries and fallopian tubes also removed.
2. • mtDNA and individual loci for tracing ancestry and disease risk analysis.
   • paternity testing is well established
3. • analyze DNA samples of learners in class; genealogy- related data.
     
     • compare results with their own cultural backgrounds.
4. • two teenage students analysed fish samples from sushi bars in NV - fingerprinting technique termed ‘DNA barcoding’
   • results showed 25% mislabeling, cheaper species replacing those advertised, some restaurants did not label (RSA meat testing cases 2013)

Question 3

Discuss the two approaches that help us understand the human genome.

Answer 3

1. Comparative genomics
   - compare the human and chimpanzee genomes
   - ask how differences between these genomes might rise to differences between the species
2. Study of human mutations
   - many mutations alter phenotypes and give clues to the functions of affected regions; many mutations cause disease
   - affected regions may encode structural proteins or enzyme or regulatory proteins or RNAs, or they may be DNA sequences that are targets of regulatory mechanisms
   - understanding the effects of such mutations illuminates human biology and, often has immediate clinical applications.

Question 4

Human vs chimpanzee:

1. genomes are about ____to understand humans focus on the ___,rather than the full 3.2 Gb(vs 2.9 Gb).
2. Express___; homologous proteins are ____. With ~ 30% proteins with no difference, average ___.

Answer 4

96% identical ,~13% Mb difference

Similar proteins, identical or very similar, 2 amino acid difference (where they are different)

Question 5

How then do humans and chimpanzees develop differently?

Answer 5

By Regulation of gene expression (Wilson and King 1975)
- 4% sequence variation make profound differences in phenotype; that suggests a chaotic system, whereby tiny perturbations can lead to large changes in subsequent trajectory; superposed on overall robustness are specific changes that extent immense leverage.

Question 6

Two ways to identify crucial sequences to study.

Answer 6

1. Effect of human vs chimpanzee mutations on functions of affected loci
2. Human mutations affecting phenotype properties lacking in chimpanzees (e.g. language); avian evolution paper - skeletal system, pulmonary structure and function, edentulism + sex and reproductive traits.

Question 7

Interesting links between genomics and language:

Answer 7

Genome-dependent component to language learning - childhood window of opportunity for easy language learning.

Language, as a biological phenomenon, links genomics with other disciplines, including neurobiology, development, medicine and anthropology - genes provide clues to the possibility of language in Neanderthals and Denisovans.

Human spoken languages have many features in common with biological species - both exhibit varying degrees of similarity and diversity, dialects are analogous to haplotypes; curtail panmyxis in a population.

Some languages have become extinct, and yet some have been rescued from extinction ( e.g. Navajo, Hebrew); endangered Khomani San language

Correlation exists between people’s native language and physical features

Clustering of genetic variation among human populations and language groups - language differences can create language barrier to gene flow.

Question 8

The human genome and medicine:

1. how to prevent disease?
2. Detection and precise diagnostics
3. Genetic counseling
4. Discovery and implementation of effective treatment
5. Tunable healthcare delivery: pharmacogenomics

Answer 8

1. • vaccines, they prime the immune system to recognize pathogens
   • understanding genetic predisposition to diseases ( Z mutant, emphysema)
   • genetic abnormalities and counseling ( Huntington’s disease)
2. • Early detection of many cancers allow simple and more successful therapy.
   • e.g leukaemia classified into 7 subtypes
   • determine subtype from gene expression patterns = better prognosis and treatment
3. Carrier status e.g. phenylketonuria (PKU)
4. • Identifying metabolic features unique to a pathogen helps to identify targets for antibacterial and antiviral agents
   • knowing the structure of a target molecule permits computer-aided drug design by molecular modeling
   • alternatively, RNA (e.g. Ebola and rhesus monkeys) ; fast mutation rates in viral genomes ( HIV = 4.1 × 10-³ per base per cell; 9.2 kb)
5. Many drugs vary in their effectiveness in different patients

Question 9

Genomics in personal identification, legal applications of DNA sequencing depend on several facts:

Answer 9

1. Genomes of all individuals are unique (even monozygotic twins)
2. Genome of each person combines maternal paternal chromosomes - familial
3. Genomes contain genes that influence recognizable features. E.g. eye color
4. Use of molecular traits to identify people and relationships
   - blood groups prove innocence but not guilt
   - MHC haplotypes (ID) vs DNA sequences (conclusive proof)

Question 10

What did Sir Alec Jeffreys and his colleagues discover when they compared the sizes of the restriction fragments of DNA samples, including human family groups?

Answer 10

• DNA fingerprinting
• gel provided a ‘barcode’ unique to each individual (different individuals different patterns, children’s DNA should comprise combination parents DNA)

Question 11

Two restriction enzymes commonly used in DNA profiling?

Answer 11

HaeIII ( blunt ends) and HinfI (sticky ends); also TaqI (sticky) = palindromes

Question 12

1. Cleaving DNA produces___?
2. When analyzing through gel electrophoresis can produce ?
3. Why do different individuals give different patterns of restriction fragment size?

Answer 12

1. Restriction fragments of variable lengths ( average, 4^n = average length)
2. Restriction map
3. One cause, is a mutation in a restriction site
   Other cause, is there may be repetitive stretch of DNA (presenting variable lengths between individuals) between restriction sites - VNTRs ; variation yields RFLPs, separated by gel electrophoresis, then Southern blotting.

Question 13

Restriction endonuclease

Answer 13

Enzyme cleaves both strands, in this case not a opposing positions, leaving dangling bits of single strand called ‘sticky ends’.

While on opposite positions, leaving ‘blunt ends’.

Question 14

Personal identification by amplification of specific regions has replaced the RFLP approach, has a disadvantage of?

The method in common use now is PCR, discuss.

Answer 14

Requiring large amounts of undegraded DNA (10-15 ng of 20000-250000 bp)

• has tiny amounts of DNA suffice to amplify even 100 bp
• target Short Tandem Repeats(STRs) - contain 2-5 bp repeated from few to dozen times.
• amplification produces 200 -500 bp fragments; loci are tested + amelogenin for gender (6 bp deletion in intron 1; X chromosome)

Question 15

Discuss mitochondrial DNA in humans.

Answer 15

• have a circular molecule; 16 568 bp long with 37 Gene’s that are densely packed in a coding region
• 13 genes encode polypeptides, 2 × rRNA (most mt proteins are nuclear encoded)
• single major non-coding region (1,122bp) - hypervariable relative to coding region = formerly (NGS) ,target area for population and forensic studies
• not useful in paternity, but maternal line

Question 16

Analysis of non-human DNA sequences include:

Answer 16

1. Proof of claims that Dolly the sheep was a clone; and testing of horses and dogs confirm breeders claim pedigree.
2. Identifying the claimed species of origin of supermarket meats and checking for commercially sold endangered species
3. Request for collection of pet DNA samples to identify dissident owners; associate hair of suspect pet with trace material found in victim

Question 17

1. What is parentage testing?
2. When 15-20 autosomal loci were tested, what were the results?
3. what happens if F and M share a VNTR length?
4. If shared by F and C (missing M)?
5. if the F and C share a rare allele?
6. What is the PI at one VNTR locus vs the CPI?
7. True/false
   Is it possible to predict certain physical traits using sequences?

Answer 17

1.- is the most common; paternity testing via VNTRs(child receives VNTRs from both parents)

2. • results are two VNTR lengths at these loci,
3. if shared by Mother, father then it’s appearance in child is uninformative.
4. • the strength of paternity inference varies with frequency of appearance in population.
5. • the associated paternity index (PI) is high,
6. is likelihood of occurence of observed genotypes if a randomly selected man from appropriate population is the biological father.
   - the CPI(combined paternity index) is the product of PI’s at all loci and is conventionally translated into POP(probability of paternity)
7. True

Question 18

1. Name some of the questions raised over the genome sequence data.
2. Name the two major national repositories of human DNA sequence information.
3. What were the concerns about NDNAD UK?

Answer 18

1. 1. Privacy issues - should data storage require each individual’s consent?
2. 2. What data to store? - only sequence or physical traits and ethnicity ?
3. 3. Access - who should be granted?
4. National DNA database- law enforcement agencies, UK BioBank - improve prevention, diagnosis and treatment of illnesses ( clinical data and biological samples to provide sequence info)
5. There have been no ethical review, abused information, extended to entire population (costs) vs criminals only (ethnic and gender bias)