17 Genetics and Development 1

Question 1

Describe general features of the human genome and the chromosomal basis of inheritance (mitosis and meiosis)

Answer 1

• 3.2 billion DNA base pairs
• 20,000 protein-encoding genes (1.5% of genome)
• Mitosis: no change in ploidy (diploid parent produces diploid daughter)
• Meiosis: Diploid parent produces four haploid gametes

Question 2

Interpret gene location using ISCN nomenclature (eg 6p21.1)

Answer 2

ISCN Nomenclature
- Numbered consecutively from centromere to telomere
- Ex 6p21.1
- Chromosome 6
- p = short arm
- Region 2
- Band 1
- Sub-band 1

ISCN = international system for Human Cytogenetic Nomenclature

Question 3

Describe basic principles of cytogenetics (karyotyping)

Answer 3

Karyotyping is the process by which a karyotype is prepared from photographs of chromosomes, in order to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities.
1. Sample collection and tissue culture.
2. Arresting cells at metaphase.
3. Swelling, separating and spreading chromosomes using hypotonic solution.
4. Separating chromosomes onto the slide.
5. Staining or banding.
6. Arranging the results- a karyotype.

Question 4

Define genetics

Answer 4

Study of genes and their roles in inheritance

Question 5

Define genomics

Answer 5

Study of all a persons genes, including interactions of those genes with the persons environment

Question 6

Define epigenetics

Answer 6

• study of reversible changes in the chromatin landscape as determinants of gene function rather than on changes of the genomic sequence itself
• ex DNA methylation, histone modifications

Epigenetic mechanisms DO NOT alter the underlyiing DNA sequence

Question 7

Define Ploidy
-Euploidy?
-Aneuplody?
-polyploid?

Answer 7

Ploidy: Number of chromosome sets in a cell
- Euploidy: normal (ie 46, XY/ 46XX)
- Aneuploidy: abnormal (ie 47, XY, +21) Male with 47 chromosomes (extra copy of 21)
- Polyploid: 3+ chromosome sets (ie triploid, tetraploid, pentaploid etc) // Not usually compatible with life (ft in cancer cells)

Question 8

Define mosaicism
How might it arise?

Answer 8

• 2 or more cell lines in an individual
• Common cause is nondisjuction in an early postzygotic mitotic division

Question 9

Contrast genetic disease with congenital disease

Answer 9

Congenital Disease
-Present at birth
-Congenital = born with
-Does not imply or exclude a genetic basis for birth defect
-1/25 babies have congenital defects (canada)
Genetic Disorder = Specific Genetic Cause
-Single gene disorders with large effects
(eg sickle cell anemia, Cystic fibrosis)
-Chromosomal disorders
(eg Trisomy 21 // Chromosome 22q11.2 deletion (digeorge syndrome))
-Complex multigenetic disorders
(Diabetes, schizophrenia, crohn disease)

Question 10

Classify genetic disorders (3 types)
Examples of each

Answer 10

1. Single-gene disorders: where a mutation affects one gene. Sickle cell anemia is an example.
2. Chromosomal disorders: where chromosomes (or parts of chromosomes) are missing or changed. Chromosomes are the structures that hold our genes. Down syndrome, DiGeorge syndrome (22q11.2)
3. Complex (multigenetic) disorders: where there are mutations in two or more genes. Often your lifestyle and environment also play a role. Diabetes, schizophrenia, crohn disease Colon cancer is an example.

Question 11

What is a GENE?

Answer 11

Basic physical unit of inheritance
Sequence of nucleotides in DNA that encodes either RNA or protein

Question 12

During which phase of cell division are chromosomes visible?

Answer 12

Metaphase chromosomes are visible

Question 13

Metaphase chromosomes are classified by (?)

Answer 13

Metaphase chromosomes are classified by the position of the centromere:
- Telocentric
- Acrocentric
- Submetacentric
- Metacentric

Question 14

What is g-banding?

Answer 14

G- banding (Giemsa banding)
-technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes

Question 15

Metaphase chromosomes are classified by the position of the centromere:
What are the four types of chromosome?

Answer 15

• Telocentric
• Acrocentric
• Submetacentric
• Metacentric

Question 16

Submetacentric chromosome

Answer 16

-centromere present near the middle and divides the chromosome into two unequal arms.
- It is an L-shaped chromosome.
Most chromosomes in humans are submetacentric, e.g. 2nd, 4th to 12th, 17th, 18th, and X chromosomes.

Question 17

Telocentric chromosome

Answer 17

-centromeres present at the end of the chromosome.
-It is not observed in humans.
-The shorter p arm is barely visible.
-The shape of the chromosome at the anaphase is like the letter ‘i’.

Question 18

Metacentric chromosome

Answer 18

-centromere separates the chromosome into two equally sized arms and is located in the center of each one.
-The metacentric chromosome is shaped like an X.
E.g. 1st, 3rd, 16th, 19th, and 20th are metacentric chromosomes in humans.

Question 19

Acrocentric Chromosome?

Answer 19

• centromere is present near the end of chromosomes.
• It forms a very short p arm and a very long q arm.
  E.g. 13th, 14th, 15th, 21st, 22nd, and Y chromosomes are acrocentric in humans.

Question 20

Limitation of karyotyping? Other tests that can be done?

Answer 20

• Cannot detect small rearrangements (<5Mb) Regions where banding is not distinctive
• Other tests: FISH , Microarray

FISH = Fluorescence in situ hydridization

Question 21

What is FISH?

Answer 21

Fluorescence in situ hydridization
- molecular cytogenetic technique that allows the localization of a specific DNA sequence or an entire chromosome in a cell.

Question 22

What is Microarray?

Answer 22

A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time.
- DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene
- can identify small duplications or deletions of genetic material that previously could not be identified using conventional karyotyping alone
- important for Microdeletion

Question 23

What is Sanger Sequencing? how does it differ from PCR?

Answer 23

Sanger sequencing, aka the “chain termination method”, is a method for determining the nucleotide sequence of DNA
- uses a DNA sequence of interest as a template for a PCR that adds modified nucleotides, called dideoxyribonucleotides (ddNTPs), to the DNA strand during the extension step
- Basic priciples of molecular assays
- Sanger sequencing differs from PCR in that only a single primer is used in the reaction.

Question 24

What is real time PCR?

Answer 24

Real-time PCR is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. This is achieved using a variety of different fluorescent chemistries that correlate PCR product concentration to fluorescence intensity (1)

Question 25

Restriction fragment length analysis?
What is an RFLP probe?

Answer 25

RFLP analysis: form of genetic testing to observe whether an individual carries a mutant gene for a disease that runs in his or her family
- An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus.
- Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.

RFLP = Restriction Fragment Length Polymorphism

uses specific restriction endonucleases

Question 26

What is Single-base primer extension?

Answer 26

Single-base extension (SBE) is a method for determining the identity of a nucleotide base at a specific position along a nucleic acid
- an effective and sensitive tool that can type over 30 known loci scattered throughout an organism’s genome in a single reaction.

Question 27

What is Next Generation Sequencing

Answer 27

Next-generation sequencing (NGS) is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed. The technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA.

Question 28

Next generation sequencing vs Sanger sequencing?
- NGS, the genetic material (DNA or RNA) is ?, to which ? of known sequences are attached, through a step known as ?, enabling the fragments to ?
The bases of each fragment are then identified by ?.
- The main difference between Sanger sequencing and 2G NGS stems from ?, with ? allowing the processing of millions of reactions in parallel, resulting in ?, ?, ? and ?

Answer 28

NGS, the genetic material (DNA or RNA) is fragmented, to which oligonucleotides of known sequences are attached, through a step known as adapter ligation, enabling the fragments to interact with the chosen sequencing system.
The bases of each fragment are then identified by their emitted signals.
The main difference between Sanger sequencing and 2G NGS stems from sequencing volume, with NGS allowing the processing of millions of reactions in parallel, resulting in high-throughput, higher sensitivity, speed and reduced cost

Question 29

Amplicon?

Answer 29

an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events

Question 30

What is Cri Du Chat Syndrome?

Answer 30

• Cat’s cry syndrome - unusual high pitched cry
• 5p deletion syndrome
• Microcephaly, hypertelorism, epicathal folds, low set ears, sometimes with preauricular tags and micrognathia
• Overal incidence estimated to be as high as 1/15000 births
• 46,XX,del5p15