EXAM 3 - The Human Genome Project

Question 1

Explain the goals of the Human Genome Project.

Answer 1

Goal: identify all the genes in the human genome
1. Create genetic linkage map - marks all the different genes with markers (1994 - few million base pairs)
2. Create physical map - street number + neighbors (1998 - sequence tagged sites)
3. Sequence all base pairs - determine which nucleotides correlate with which genes (2003)
4. Create tools, forums & training; find disease predictors.

Question 2

What was the final price of the Human Genome Project?

Answer 2

$5 billion

Question 3

Explain why Venter created Celera Genomics?

Answer 3

• He thought that the HGP was too slow and too expensive
• Goal: faster and 10x cheaper
• Celera stocks plummented bc human genomes cannot be patented

Question 4

Describe the benefits of the HGP.

Answer 4

• All human genes have been identified and annotated sequences are stored in free public databases - where the gene starts and ends
• Provides prenatal and presymptomatic diagnoses of disorders in individuals that can carry disease gene
• Consortiums are organized which store cDNA collections for identified genes –> publicly available for a fee
• Rapid development of cost efficient sequencing technology
• Computers can visualize and search the databases
• Ethical, legal, and social implications have been examined

Question 5

Describe Maxam-Gilbert sequencing.

Answer 5

In 1976-1977, Maxam and Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases.

Question 6

Explain what is required for Chain-termination methods.

Answer 6

AKA Sanger PCR-based
Required:
* a ssDNA template
* DNA primer
* a DNA polymerase
* radioactively or fluorescently labeled nucleotides
* modified nucleotides that terminate DNA strand elongation

Question 7

Explain the function of ddNTPs on chain-termination.

Answer 7

DNA elongation inhibitors
* cause termination when inserted
* do not have hydroxyl group on C3

Question 8

Explain automated sequencing.

Answer 8

Capillary electrophoresis + fluorescently labeled ddNTPs
* production of automatic sequencing machine

Question 9

Describe the process of chain-termination methods.

Answer 9

How do we determine the sequence of the entire genome?
1. DNA extraction
2. DNA fragmentation
3. Clone into vectors
4. Transform bacteria, grow, isolate vector DNA
5. Sequence the library
6. Assemble continuous fragments

Question 10

Explain the 3rd generation sequencing machine.

Answer 10

We can detect the nucleotide sequence during elongation through fluorescence
* DNA polymerase uses fluorescently tagged bases to synthesize DNA.
* Each base has a specific color that flashes as it gets added to the DNA strand.
* Pacific Biosciences baosted that instruments would be able to sequence a human genome in just 15 mins by 2013

Question 11

Describe the types of Next Generation Sequencing.

Answer 11

• Illumina sequencing
• Roche 454 sequencing
• Ion torrent: proton/PGM sequencing
• SOLID sequencing

Question 12

Explain the process of NGS sequencing.

Question 13

What are the 4 main advantages of NGS over chain-termination methods?

Answer 13

• speed
• cost
• sample size
• accuracy

Question 14

Explain (4th gen.) nanopore sequencing.

Answer 14

MinION and SmidgION
* A strand of DNA is passed through a nanopore. The current is changed as the bases G, A, T and C pass through the pore in different combinations.

Question 15

How are human genes categorized?

Answer 15

Categorized by function of the transcribed proteins, given as both number of encoding genes and percent of all genes.
* some noncoding DNA contain genes for RNA molecules with important biological functions.

Question 16

Explain SNPs.

Answer 16

Single nucleotide polymorphisms
synonymous
AAG –> AGG = arg –> arg

non-synonymous
CGG –> GGG = arg –> gly
* possible consequences on protein function
* PK drug metabolism
* response to drug may be different

CGA –> TGA = arg –> STP
* truncated protein less stable

Question 17

What is the difference between SNPs and mutations?

Answer 17

SNP - type of mutation that occurs in a single nucleotide in the genome
Mutation - can be many types of changes in the structure/variation in the DNA sequence.
* found in small fraction of population
* source for genetic disease

Question 18

What are some examples fo emerging genomic medicine in action?

Answer 18

• cancer
• pharmacogenomics
• rare genetic disease diagnostics
• non-invastice prenatal screening
• clinical genomics info system

Question 19

Explain the goals of the Earth BioGenome Project (EBP).

Answer 19

For the first time in history, it is possible to efficiently sequence the
genomes of all known species
Sequence, catalog, and characterize the genomes of all of Earth’s eukaryotic biodiversity over a period of 10 years.
* Goal: create a new foundation for biology to drive solutions for preserving biodiversity and sustaining human societies.

Question 20

Describe the difference between epigenetics and epigenomics.

Answer 20

Epigenetics - study of changes in the regulation fo gene activity and expression that are not dependent on gene sequence.
* studies process that regulate how and when certain genes are turned on/off
* heritable changes and also stable, long-term alterations that are not necessarily inheritable

Epigenomics - global analysis of epigenetic changes across the entire genome.

Question 21

Explain the challenges in analytic methods of proteomics.

*

Answer 21

• Complexity of protein structure
• Protein isolation/identification / Abundance (Lack)
• Localization of action in tissues/ Tissue heterogeneity
• Post-translational modification / Isoforms
• Protein properties – mass, isoelectric point, hydrophobicity
• Function determination in health & disease
• Interactions of proteins with other biomolecules (Yeast two-hybrid system)
• Coordinating information from multiple tests/Informatics

Question 22

Explain the yeast two-hybrid system.

Answer 22

• 2 domains: binding domain and activaiton domain
• The two domains must bind to the promoter –> activate transcription
• Bind protein X to binding domain and bind protein y to activation domain –> increase transcription

Question 23

Describe the process of proteomic protein identification.

Answer 23

• Sample preparation
• first dimension isoelectric focusing
• SDS-PAGE - protein staining
• image analysis –> you can tell which protein is important for development of a specific disease by identifying which is missing/present in the disease versus normal state
• mass spectrometry –> protein identification

Question 24

Describe the process of proteomic protein identification.

Answer 24

• Sample preparation
• first dimension isoelectric focusing
• SDS-PAGE - protein staining
• image analysis –> you can tell which protein is important for development of a specific disease by identifying which is missing/present in the disease versus normal state
• mass spectrometry –> protein identification