Genomics

Question 1

What are the types of genomics?

Answer 1

Genome: the entire genetic compliment of an organism, encoded in nucleic acids

Note: Most genomes are made of DNA (deoxyribonucleic acid) but a few viruses have RNA

Prokaryotic genomes:
-DNA concentrated in nucleoid

-Extrachromosomal as plasmid DNA

Eukaryotic genomes
-Nuclear genomes

• Mitochondrial genomes
• Chloroplast genomes

Question 2

What are cytogenetics?

Answer 2

Diagnostics of chromosomal abnormalities

Karyotype analysis detects:

Euploidy

Aneuploidy

Structural Chromosomal abnormalities

Question 3

What is euploidy?

Answer 3

The normal diploid complement of 46 chromosomes, 22 autosome pairs and two sex chromosomes (46, XX or 46, XY)

Question 4

What is aneuploidy?

Answer 4

The number of chromosomes is different from normal

• Only aneuploidy that are compatible with life are:
  
  • Extra chromosomes 21,18, or 13(e.g., 47, XY, +21)
  • Extra X or Y chromosome (e.g., 47, XXY, Klinefelter syndrome)
  • Single X chromosome(Turner syndrome)

Question 5

Describe the cryogenic map

Answer 5

The nuclear DNA is densely packaged into chromosomes

• The characteristic banding pattern (represented by an ideograms) of each chromosome is the basis of the cytogenetic map of the genome
• The banding comes from a stain called Geimsa

Question 6

What is a karyotype?

Answer 6

Chromosome organized by a cytogenicist

Normal female- 46, XX,

Normal male- 46, XY

Question 7

What is the purpose of Fluorescence in situ hybridization (FISH)?

Answer 7

This technique allows the localization of large DNA fragments on metaphase chromosomes or interphase chromatin strands

-A DNA fragment is labelled (typically radioactive or fluorescent) and hybridized to metaphase chromosomes

Whole chromosome specific DNA probes
Whole chromosome paints can be used to sort and isolate specific chromosomes

Question 8

What is the purpose of Standard Karyotype Analysis?

Answer 8

Has been used for some time to test for major chromosomal abnormalities. However, it is unable to resolve micro deletions

Question 9

What is the purpose of the aneuploidy screen test?

Answer 9

-Shows a female fetus with trisomy-21

• the nucleus on the right has been hybridized to fluorescent probes for chromosomes
  
  • 13 (green) and
  • 21(red) clearly has three red signals

Question 10

What is a micro deletion?

Answer 10

A microdeletion would be the deletion of one, a few, hundreds, thousands or up to 3 million base pairs

Question 11

Explain detecting microdeletions by FISH

Answer 11

Microdeletions may be too small to be detected by light microscopy using standard karyotyping or aneuuploid screen test

This is an example of a metaphase cell that has been hybridized with the probe for Steroid Sulfatase Deficiency which is caused by a microdeletion on the X chromosome

• The “X cen” probe signal is an internal control and is located at the X chromosome
• Since there are two X chromosome and only one has the Steroid Sulfatase gene signal, this individual is a female carrier for Steroid Sulfatase Deficiency

Question 12

Explain identification of specific DNA sequences within a genome

Answer 12

Restriction Fragment length polymorphism (RFLP) and Southern blot analysis

1-2: genomic DNA is isolated and digested with a restriction enzyme

3: the resulting cut DNA is separated by gel electrophoresis
4: The DNA fragments are denatured and transferred to a membrane (Southern blotting) and
5: the membrane is hybridized with a labeled DNA fragment corresponding to the chromosomal region containing the polymorphism

Question 13

Describe restriction sites on B-globin gene

Answer 13

Cleavage of the gene for B-globin with a restriction endonuclease

Restriction site present in normal DNA and sickle cell DNA
-Restriction site present in normal DNA but missing in sickle cell DNA

Electrophoresis of restriction fragments from the DNA of normal individuals yields a 1,150 bp fragment using a probe specific for the B-globin gene

Electrophoresis of restriction fragments from the DNA of a patient with sickle cell disease yields a 1,350 bp fragment because of the loss of a cleavage site

-Electrophoresis of restriction fragments from the DNA of a heterozygote yields both 1,150 bp and 1,350 bp fragments

Question 14

What are the functions of Allele Specific Oligonucleotide(ASO) Probes & SNPs?

Answer 14

• a DNA marker resulting from a base pair difference at one particular site in a genome
• alleles of an SNP can be typed by using oligonucleotide hybridization analysis
• a short oligonucleotide is synthesized that is complementary to the allele at the SNP locus. The oligonucleotide is hybridized under high stringency (high temperature, low salt) to DNA samples spotted on membranes

Question 15

What is Sanger (dideoxy) sequencing?

Answer 15

Method developed in the 1970s by Frederick Sanger

-to determine the exact order of nucleotides in a given DNA or RNA sequence & to see smaller changes in whole genomes

Sanger won the Nobel prize in chemistry 1980 for sequencing technology

Extension (synthesis) of a DNA strand requires a 3’-OH group. This is absent from deoxynucleotides

Question 16

What is the principle of Sanger sequencing?

Answer 16

Based on extending a DNA strand from a primer that is bound to a ssDNA template

• Based on the termination of DNA chain extension by a deoxynucleotide (lacks a 3’- hydroxyl group (e.g., ddTTP)
• The reaction contains both deoxynucleotides and labelled dideoxynucleotides (as well as DNA polymerase, a complementary primer, and buffers)
• the random incorporation of a deoxynucleotide terminates the synthesis of the DNA strand
• Four reactions are conducted each containing a different dideoxyribonucleotide

Question 17

What is the impact of being able to fluorescently label nucleotides ?

Answer 17

Traditionally, nucleotides were radioactively labeled and each reaction (A,C,G,T) were conducted separately and run on a gel separately

The development of fluorescent labeled nucleotides meant that all reactions could be conducted in a single tube, run on a single lane of a gel and automated

Question 18

Explain the functioning of computer automated DNA sequencing

Answer 18

1. Reaction components:
   - DNA template
   - Primer
   - DNA polymerase
   - dNTPs
   - small amount of ddNTPs with fluorochromes regular DNA bases-90%, ddNTPs-10%
2. Primer extension and chain termination
3. Separation DNA fragments by capillary gel electrophoresis
4. Laser and detector detect fluorescence of each ddNTP and provide input to a computer for sequence analysis

Question 19

What is the physical clone map?

Answer 19

Individual clones are assembled into contigs and a representive overlapping set (the titlting path) is selected for sequencing and further studies

Question 20

How is a clone map integrated with a cytogenic map?

Answer 20

Clones selected from the map are fluorescently labelled and localized to chromosomes to integrate the physical clone map with the cryogenic map

• A library is constructed by fragmenting the target genome
• cloning genome into a large-fragment cloning vector; here, BAC vectors are shown
• The genomic DNA fragments represented in the library are then organized into a physical map
• individual BAC clones are selected and sequenced by the random shotgun strategy
• Finally, the clone sequences are assembled to re-construct the sequence of the genome

Question 21

Describe the major events of exploring the human genome

Answer 21

1. Sanger sequencing, targeted genotyping
2. Genome-wide genotyping(GWAS)
3. Exome sequencing
4. Genome sequencing

Human genome project—> international hapmap project
—> 1000 genomes- a deep catalog of human genetic variation

Question 22

What are the goals of the human genome project?

Answer 22

Goals- identify all the genes in human DNA

• determine the sequences of the 3 billion nucleotides that make up human DNA
• Store this information in publicly accessible databases

Methodology- DNA samples were collected from several anonymous volunteers

• DNA was distributed to genome centers around the world
• Researches performed mapping, sequencing, and analysis using specialized equipment
• Sequences were placed into a central database

Question 23

Describe the human genome projects

Answer 23

In June 26 2000, at The White House, it was announced that the a human Genome Project was essentially completed by:

• Celera Genomics (private company -$300 million)
• The National Human Genome Research Initiative and Its international Partners(publicly funded- $2.7 Billion)

Craig Venter(Celera)

Francis Collins(NIH)

Question 24

What is haplotype?

Answer 24

Combination of alleles at adjacent locations on a chromosome that are inherited together

Question 25

What is the HapMap?

Answer 25

The Hapmap is a catalog of common genetic variants that occur in human beings - It describes what these variants are - Where they occur in our DNA - how they are distributed among people within populations and among populations in different parts of the world. The Project is designed to provided information that other researchers can use to link genetic variants to the risk for specific illnesses, which will lead to new methods for specific illnesses, which will lead to new methods of preventing, diagnosing, and treating disease

Question 26

What is the 1000 Genome Project?

Answer 26

Whole genome sequencing and complete description of human genetic diversity in >1000 individuals from multiple world populations The goal of the 1000 Genome Project is to find most genetic variants that have frequencies of at least 1% in the populations studied

Question 27

What is a microarray?

Answer 27

A microarray is a collection or a grid of microscopic spots of biological capture molecules (like DNA of proteins) which can be probed with target molecules like DNA, RNA, antibodies or proteins

Question 28

What are probes?

Answer 28

DNA microarrays (DNA chips or bio chips): each spot contains predefined short (25- to 70-nucleotide) single-stranded DNA oligonucleotide or larger (200- to 800- basepair) double-stranded DNA, called probes - Comparative Genome Hybridization (CGH) - SNP chips - expression arrays - Tiling Arrays

Question 29

Describe Comparative Genome Hybridization (CGH)

Answer 29

-Comparing a patient DNA to Control DNA looking for missing regions or duplicated regions (insertions or deletions) - Copy number variations (CNV) - Each person should have 2 copies of every gene on autosomes (not sex chromosomes) - Some people have more or less copies of DNA regions - Comparing control to tumor DNA

Question 30

What are the steps in Array CGH?

Answer 30

Steps 1-3: Patient and control DNA are labeled with fluorescent dyes and applied to the microarray Steps 4: Patient and control DNA compete to attach, or hybridize, to the microarray Steps 5: The microarray scanner measures the fluorescent signals Steps 6: Computer software analyzes the data and generates a plot

Question 31

Describe Single Nucleotide Polymorphism (SNP) CHIPS

Answer 31

- Looking for all the unique single base pair changes that are known - a DNA marker resulting from a base pair difference at one particular site in a genome - alleles of an SNP can be typed by using oligonucleotide hybridization analysis - a short oligonucleotide is synthesized that is complementary to the common allele at the SNP locus. The oligonucleotide is hybridized under high stringency (high temperature, low salt) to DNA samples spotted on membranes - the oligonucleotide will not hybridize with target DNA that has the single base pair change

Question 32

What do SNP microarray (SNP chips) involve?

Answer 32

Analysis involves labeling genomic DNA fragments and allowing them to hybridizeDNA spots adhered onto the microarray slide Single nucleotide polymorphism(SNP)- a polymorphism where the alleles vary within one single nucleotide base Hybridization: annealing of complementary ssDNA ssDNA probe labeled with 32P or fluorescent molecule The SNP array is then scanned using a laser-microscope system to visualize which array features (gene spots) our labeled genomic DNA has hybridized to A positive signal indicates the presence of a particular SNP within the genome of that particular individual

Question 33

What is the function of next-generation sequencing?

Answer 33

Next generation sequencing(NGS) platforms carry out massively parallel sequencing -millions of DNA fragments are sequenced in unison - Rapid (sequencing an entire genome in less than one day) - Low cost in comparison to traditional techniques (Sanger sequencing)

Question 34

What are the applications of NGS?

Answer 34

- Whole-genome sequencing - of different species for comparative biology analyses - to identify genes and regulatory elements involved in disease - Whole exome-sequencing - The exome consists of only the protein-coding regions of the genome (a little over 1% of the genome) - Used in gene discovery research - For discovery of disease-causing mutations - Targeted sequencing - specifically targets regions of the genome that are of interest to researchers or clinicians - more affordable and yields much higher coverage of genomic regions of interest - Sequencing panels can be developed to target specific genomic regions or disease-causing mutation hotspots

Question 35

What are the benefits of NGS to patients?

Answer 35

Variants in individuals DNA vs ‘reference’ sequence may be valuable in: -identifying new disease genes: high diagnostic yield -identifying rare mutations for “resilience” - Providing better treatment options - preventative treatments - personalized drug regimen Differentiating disease subtypes

Question 36

Give all the examples of genetic and genomic testing in personalized medicine

Answer 36

Pre-symptomatic risk assessment -BRCA1/2 testing for Brest cancer Testing for hereditary colon cancer Diagnosis - Beta thalassemia - Fusion genes and rearrangements in pediatric leukemia Prognosis - Fragile X syndrome(number of trinucleotide repeats predict severity ) - Gene expression analysis and lymphoma prognosis Treatment and pharmacogenics -Therapies for targeted gene mutations in cancer Pharmacogenics applications - Rs2395029 testing for HLA-B*5701 allele, correlated with hypersensitivity to abacavir treatment for HIV+ patients

Question 37

What are the challenges to personalized genomics ?

Answer 37

Ethical, Legal and Social Implicstions (ELSI) -Research Program: established in 1990 as an integral part of the Human Genome Project(HGP) to foster basic and applied research on the ethical, legal and social implications of genetic and genomic research for individuals, families and communities - Privacy - Comfidentiality - Discrimination (GINA in 2008-USA) - Psychological Impact