Medical Biotechnology 1

How is fluorescence in situ hybridization (FISH) done?

A chromosome spread is prepared on a slide and then fluorescent probes are hybridized to each chromosome. Each probe is specific for certain 'marker' sequences on each chromosome. In some cases, FISH can be performed with probes that fluoresce different colors (a procedure called spectral karyotyping)

How is Amniocentesis performed?

A needle is inserted into the mother's abdomen into the pocket of amniotic fluid. Isolated cells are then cultured for a few days to increase cell numbers. Cells are then treated to arrest them in mitosis, where the mitotic chromosomes are spread onto a glass slide. The chromosomes are stained with different dyes that bind to proteins attached to the DNA, creating patterns of alternating light and dark band on each chromosome. Karyotyping is performed.

How many genes can a single microarray carry?

A single microarray can contain probes for thousands of genes.

How is chorionic villus sampling carried out?

A suction tube is used to remove a small portion of the chorionic villus.

What are ASOs?

ASOs are small single-stranded oligonucleotide sequences, usually around 20 nucleotides in length.

What is karyotyping

Aligning chromosomes into pairs based on their size and their banding pattern.

What are the methods to carry out genetic testing?

Amniocentesis. Chorionic villus sampling.

What do ASOs do?

An ASO will hybridize to a normal gene and another ASO for the mutant gene are used.

How does the body know where to develop certain organs and determine which cells must die to make room for others?

Apoptosis.

What is the disadvantage of chorionic villus sampling?

Carries higher risk than amniocentesis of disturbing the fetus and causing a miscarriage.

What are haplotypes (Hap)?

Cluster of SNPs on the same chromosome

What are the symptoms of Down syndrome (trisomy 21)?

Cognitive impairments Short stature Broadened facial features.

Where is RFLP analysis used?

DNA fingerprinting.

How is allele-specific oligonucleotide (ASO) analysis performed?

DNA is isolated from human cells, usually white blood cells, and then amplified by the polymerase chain reaction (PCR) using primers that flank a disease gene of interest. Amplified DNA is then blotted onto nylon filters and hybridized separately to two different ASOs as probes.

How is microarry analysis performed?

DNA or RNA is isolated from a patient's tissue sample. The patient's DNA is tagged with fluorescent dyes and then hybridized to the chip. Spots on the microarray where the patient's DNA bound are revealed by fluorescence.

How is RFLP analysis carried out?

Defective gene sequences may be cut differently by restriction enzymes than their normal complements because nucleotide changes in the mutant genes can affect restriction enzyme cutting sites to create more or fewer.

What does microarray analysis of cancer patients allow?

Design treatment strategies to combat cancer based on subtle differences in the expression of cancer-causing genes.

What is the advantage of Allele-specific oligonucleotide (ASO) analysis over RFLP analysis

Detect a single nucleotide change in a gene even if the mutation does not change a restriction site.

How can you detect translocation?

Detect by FISH using different colored fluorescent probes for each chromosome.

What can serve as biomarkers of disease other than e.g. PSA

Detecting individual genes or gene expression patterns.

What does elevated levels of prostate-specific antigen indicate?

Elevated levels of PSA can be a marker for prostate inflammation and even prostate cancer.

What is the advantage of chorionic villus sampling over amniocentesis?

Enough cells are obtained so the sample can immediately be used for karyotyping. Can be done earlier in the pregnancy (8-10 weeks)

What are the problems with using animal models

Ethics. Costy: each animal can cost over $50,000. Available in limited numbers. No guarantee they have the same reaction as human.

Why can we use model organisms to study human disease conditions

Genes are highly conserved. We share large numbers of genes with other organisms. A number of genetic disease also occur in model organisms.

What are protein microarrays?

Hundreds or thousands of antibodies spotted on the chip. By applying blood proteins from a patient, researchers have been able to detect illness indicated by the presence of proteins from disease-causing organisms.

What is the purpose of detecting Down Syndrome.

If detected defect, genetic tests can give parents choice to determine if they want abortion, or provide information that can be used to treat fetuses during pregnancy and after the child is born.

Where are biomarkers released

Many biomarkers are released into body fluids such as blood and urine as a product of cell damage.

What is a model organism for Down syndrome?

Mice with almost a complete copy of human chromosome 21. These mice show characteristics of Down Syndrome and may turn out to be a very valuable model for understanding the genetics of this condition.

What can be currently identified from microarrays?

Microarrays are currently being used to identify genetic differences in patients with various types of cancer.

What is microarrays used for?

Microarrays may be used to screen a patient for a pattern of genes that might be expressed in a particular disease condition. Microarray data can then be used to predict the patient's risk of developing disease based on the patient's expressed genes for the disease.

Why don't most SNPs have an effect on cells?

Most SNPs occur in non-protein coding regions (introns) of the genome.

What genetic conditions do genetic tests focus on?

Most of the genetic testing focus on genetic conditions that occur as a result of alterations in chromosome number or large structural abnormalities of chromosomes.

What cause the release of prostate-specific antigen (PSA)

PSA is released into the bloodstream when the prostate gland is inflamed.

When is Amniocentesis carried out?

Performed when the developing fetus is around 16 weeks of age.

What are the questions raised about microarray analysis?

Questions raised about the importance of quality control and consistency measures in using microarrays for genetic testing.

Give an example of gene knockout

Scientist discovered that mice without Ob gene can become obese. Ob codes for leptin hormone, which is used to regulate hunger. Discovered human homologue for leptin provide insight into fat metabolism in humans and the genetics that may influence weight disorders. Extremely obese children in England have been treated with leptin and have responded very well in preliminary studies.

What was the outcome from the Human Genome Project?

Scientists developed maps showing the locations of normal and diseased genes on all human chromosomes.

What follows the HGP?

The HGP has led to the development of The Cancer Genome Atlas Project (TCGA).

What can you tell from a karyotype

The sex based on the prescence of the sex chromosomes (X, Y). Identify if there is abnormal chromosomal numbers.

During Amniocentesis, why are cells arrested in mitosis

To facilitate the viewing of mitotic chromosomes

Give an example of a genetic disease due to alterations in chromosome number.

Trisomy 21

What are the advantages of FISH

Very useful for identifying missing chromosomes and extra chromosomes. Makes it much easier than conventional karyotyping to detect defective chromosomes

What is translocation

When a portion of a chromosome is deleted or a piece of chromosome is swapped from one chromosome to another due to problems in chromosomal replication.

What is The Cancer Genome Atlas Project?

a comprehensive effort to identify genomic changes involved in a variety of different cancers (identify what trigger normal cells to become cancerous cells).

What is the purpose of preimplantation genetic testing?

allow individuals to select a healthy embryo prior to implantation.

What is the disadvantages of RFLP analysis?

can only be used to analyze gene defects in which mutation changes a restriction site in a gene.

What does the amniotic fluid contain

contains cells shed from the fetus, such as skin cells.

What is the purpose of Restriction fragment length polymorphism (RFLP) analysis

detect genetic diseases in embryos and adults from either amniotic cells or blood cells.

What is the importance of biomarkers

early detection and disease diagnosis.

What is the purpose of gene knockout?

eliminate a gene that is thought to play a role in human illness in model organisms. Study the effects on the organism to learn about the functions of the gene.

What is the function of chorionic villus

fetal tissue that helps form the placenta.

Why is RFLP analysis called 'restriction fragment length polymorphism'?

fragments of different length or forms created by restriction enzymes.

What can scientist do with model organisms

identify disease genes and test gene therapy and drug-based therapeutic approaches to evaluate their effectiveness and safety in preclinical studies before using them for clinical trials in humans.

What are most genetic tests on fetus for?

identifying sex of the child or detecting small numbers of genetic diseases.

What does the binding of a patient's DNA to a gene sequence on the chip indicate?

indicates that his or her DNA has a particular mutation or SNP.

What happens when a SNP occur in a gene sequence?

it may cause a change in protein structure that produces disease or influences traits in a variety of ways, including conferring susceptibility for some types of disease conditions.

What are model organisms

mice, rats, worms, flies. Help scientists study human disease conditions.

What is a Noninvasive prenatal genetic diagnosis (NIPD) test?

non-invasive fetal test based on identifying and sequencing small amounts of fetal chromosomal fragments present in a pregnant woman's blood.

What is Single Nucleotide Polymorphisms (SNPs)

nucleotide changes in DNA sequences that vary from individual to individual.

How frequent is SNPs in human genome?

occur approx. every 100-300 base pairs in the human genome.

What is apoptosis

programmed cell death.

What are biomarkers

proteins released by dead and dying cells such as cells undergoing apoptosis.

What does SNPs tell you?

represent one of the most common forms of genetic variation among humans. Estimated that SNPs make up abotu 90% of human genetic variation.

What is meant by homologous

similar DNA sequence in model species and human

What is the function of amniotic fluid

surround and cushion the fetus.

What is the purpose of the HapMap Project

to identify and catelogue the chromosomal locations (loci) of more than 1.4 million SNPs that are present in the 3 billion bp of the human genome and to understand the roles of SNPs in disease diagnosis and treatment.

What is the use of PCR, ASO analysis, FISH?

to screen for gene defects in single cells from 8- to 32- cell embryos created by in vitro fertilization.

What are some SNPs used for?

used to predict susceptibilities to ailments such as stroke, diabetes, cancer, heart, disease, behavioural and emotional illnesses, and a host of other disorders that may have a genetic basis.