Project Lead The WayMedical Intervention

Exam 2 Review

Disclaimer

This review is an overview. There is no possible way to revisit every single item we have covered thus far. This should be used as 1 tool in your preparation for the exam.

Major Focus

Key Terms – Crossword PuzzlesKey Concepts – Each Activity/ProjectEssential Questions – Each UnitConclusion Questions – Each Activity/ProjectKnowledge and Skills – Each Unit

Exam 2 Review

Unit 1.4.1 through Unit 2.2.2 1.4.1.A Vaccinations 1.4.2 Types of Vaccines 1.4.2.A Vaccine Development 1.4.3.A Epidemiologist

2.1.1.A Genetic Counseling 2.1.3.A Test Genes 2.1.5.A Fetal Health 2.2.1.A Gene Therapy 2.2.2.A Reproductive Tech

Unit 1.4

Vaccinations

Unit 1.4 - Overview

In the first activity of the lesson, you will study the history of vaccination through the eyes of scientist Edward Jenner. Smallpox, a highly infectious disease characterized by small lesions on the skin, ravaged society for centuries.

Edward Jenner• English physician/scientist who pioneered smallpox vaccine - the world's first

vaccine• In 1796 - Jenner inoculated James Phipps, an 8 year old boy • He scraped pus from cowpox blisters on the hands of a milkmaid who had

contracted cowpox and injected in both arms of Phipps• Phipps presented with a fever and some uneasiness, but no full-blown infection• Testing showed he developed an immunity to smallpox

What is a vaccination and how does it work?

The body is presented a dead or weakened form of the pathogen to expose the immune system to the antigen.

This allows the B-cell to produce antibodies and memory cells.

Therefore, when the body is exposed to the antigen again the immune system will be able to fight off the infection.

How has vaccination impacted disease trends in our country?

Herd Immunity

• More individuals that are immune decreases the incidence of the disease and the occurrence of the pathogen.

• With greater numbers immunized, it is less likely that an unimmunized person will encounter the pathogen.

• Mass vaccination confers indirect protection for those who do not receive the vaccine resulting in “herd immunity”.

Effective Vaccines

• Have low levels of side effects or toxicity.• Protect against exposure to natural, or wild forms of the

pathogen.• Should stimulate both an antibody (B-cell) response and

a cell mediated (T-cell) response.• Have long term, lasting effects that produce

immunological memory.• Should not require numerous doses or boosters• Are inexpensive, have a long shelf life and are easy to

administer.

Routes of Administration

• The majority of vaccines are administered by injection– Subcutaneous– Intramuscular– Intradermal

• Oral vaccines are available for only a few diseases

Methods Used to Produce Vaccines in the Laboratory?

1) Killed2) Attenuated3) Toxoid4) Subunit – recombinant DNA technology5) Naked DNA6) Similar Pathogen

2 goals for every vaccine– Contains Antigen so we can produce antibodies– Pathogen will not be harmful

Types of Vaccines

Killed whole cells or inactivated viruses– Pathogen is killed due to heat or radiation and inserted into

the body– Even though they are harmless, they still contain

recognizable antigens on their surface– Because the microbe does not multiply, larger doses and

more boosters are required.

Types of Vaccines

Live, attenuated (weakened) cells or viruses– Pathogen is grown under non ideal conditions for several

generations, causing the pathogen to evolve – Due to natural selection the pathogen is now adapted to the

new environment – When placed in humans they still have the antigens, but will

be weak in the body’s environment so they do no harm– Longer-lasting and require fewer boosters– Disease agent could mutate back to pathogenic strain

Types of VaccinesToxoid vaccines

– Grow pathogen and collect the toxins produced by the antigen

– Purified toxin injected into the person with another vaccine and the body will elicit immune response

Genetically engineered – Recombinant DNA Tech– Genes for microbial antigens are inserted into a plasmid

vector and are cloned in appropriate hosts– The resultant protein product is used to provoke immune

system

DNA vaccines – Naked DNA– These vaccines contain all or part of the pathogen DNA,

which is used to “infect” a recipient’s cells

Subunit Vaccine

Gene for antigen-only from pathogen is put onto a plasmid and inserted into another organism. This organism can produce the antigen, which is used to produce the vaccine.

What is recombinant DNA technology?

joining together of DNA molecules (from two different species) that are inserted into a host organism to produce

new genetic combinations that are of value to science, medicine, agriculture, and industry.

What are the molecular tools used to assemble recombinant DNA?

• Restriction Enzymes cut DNA and open Plasmids• Ligase connects DNA fragments to a plasmid at the sticky ends• This forms a recombinant plasmid that has a specific gene in it

recombinant DNA

technology

Inserting engineered plasmids into bacterial

cells

• The recombinant plasmid is made and inserted into the bacteria via transformation.

• The bacteria can express the gene that was placed into the plasmid to produce the necessary antigen for the vaccine.

Similar Pathogen Vaccine

Insert a similar pathogen to what you want to vaccinate against. This pathogen is not as harmful to humans as the pathogen you are vaccinating against. It contains a similar enough antigen that we can develop antibodies to kill the pathogen.

Small Pox – Cow Pox

Naked DNA Vaccine

Gene for antigen from pathogen is put onto a plasmid and inserted into a bacteria to replicate (copy) the plasmid. The plasmid is purified and placed into the body.

The body cells can uptake the DNA and begin making the antigen.

Immune ResponseWhite Blood Cells (WBC) are the primary cells responsible for an immune response

WBC are either (1) myeloid leukocytes or (2) lymphocytes

Cells of the myeloid lineage include neutrophils, monocytes, eosinophils and basophils.

Lymphocytes include T (thymus) cells, B (bone marrow) cells and natural killer cells.

Lymphocytes start out in the bone marrow and either stay there and mature into B cells, or they leave for the thymus gland, where they mature into T cells.

B lymphocytes and T lymphocytes have separate functions: • B lymphocytes seek out (recognize) their targets • T cells destroy the invaders

Memory B cells – float around seeking 1 specific antigen – concentration varies

B Cell T Cell

What is epidemiology?

Epidemiology is the study of the spread, cause, and effects of diseases in certain populations.

How can epidemiologists assist with the detection, prevention, and

treatment of both chronic and infectious disease?

• They analyze data, conduct surveys, and perform tests, to identify the cause and spread of the disease.

• They develop informative tools and use preventative measures to stop the spread of the disease.

2.1.1

Genetic Disorders and Genetic Testing

© 2010 Project Lead The Way, Inc.Medical Interventions

What are Genetic Disorders?

• Both environmental and genetic factors play a role in the development of disease.

• A genetic disorder is a disease caused by abnormalities in an individual’s genetic material.

Four different types of genetic disorders:• Single-gene• Multifactorial• Chromosomal• Mitochondrial

Single Gene Disorders

• Single gene disorders are caused by changes or mutations that occur in the DNA sequence of one gene.

• Remember that a gene, a segment of DNA, contains instructions for the production of a protein.

Mutated DNA = Mutated protein!!

• Diseases and disorders result when a gene is mutated resulting in a protein product that can no longer carry out its normal job.

Single Gene Disorders• Single gene disorders are inherited in

recognizable patterns:– Autosomal dominant– Autosomal recessive – Sex linked

• Genetic testing looks at genotype to determine if someone has a genetic disorder, will develop one, or is a carrier.

Autosome = any chromosome other than sex chromosome

Autosomal recessive disorder means two copies of an abnormal gene must be present in order for the disease or trait to develop.

Autosomal dominant means you only need to get the abnormal gene from one parent in order for you to inherit the disease.

Multifactorial Disorders• Multifactorial disorders are caused by a combination

of environmental factors and mutations in multiple genes.– Development of heart disease is associated with multiple

genes, as well as lifestyle and environmental factors.– Different genes that influence breast cancer development

have been found on chromosomes 6, 11, 13, 14, 15, 17 & 22.

• Many of the most common chronic illnesses are multifactorial.

Chromosomal Disorders

• Humans have 46 chromosomes in their body cells.– 44 autosomes (22 pairs)– 2 sex chromosomes (1 pair)

• Because chromosomes carry genetic information, problems arise when there are missing or extra copies of genes, or breaks, deletions or rejoinings of chromosomes.

• Karyotypes, pictures of the paired chromosomes of an individual, are important in diagnosing chromosomal disorders.

Mitochondrial Disorders

• Mitochondria, the organelles in your cells that convert energy, also contain DNA.

• A mitochondrial disorder, a relatively rare type of genetic disorder is caused by mutations in nonchromosomal DNA of mitochondria.

• Mitochondiral DNA is unique in that it is passed solely from mother to child

Duchenne Muscular Dystrophy

Cystic Fibrosis

Huntington’s Disease

Down Syndrome

Leber hereditary optic neuropathy

Alzheimer’s Disease

Autosomal – both parents

Autosomal – one parent

Sex chromosome

Multiple factors influence

Chromosomal deviations

mtDNA deviations

Types of Genetic Testing and Screening

Carrier Screening• Carrier screening determines whether an individual

carries a copy of an altered gene for a particular recessive disease even though they do not show the trait phenotypically.

• Carrier screening is often used if a particular disease is common in a couple’s ethnic background or if there is a family history of the disease.

• Examples of carrier tests include those for Tay-Sachs disease or sickle cell disease.

Preimplantation Genetic Diagnosis (PGD)

• PGD is used following in vitro fertilization to diagnose a genetic disease or condition before the embryo is implanted in the uterus.

• A single cell is removed from an embryo and examined for chromosome abnormalities or genetic changes.

• Parents and doctors can then choose which embryos to implant.

The Process of Preimplantation Genetic Diagnosis

Fetal Screening/Prenatal Diagnosis

• Prenatal diagnosis allows parents to diagnose a genetic condition in their developing fetus.

• Techniques such as amniocentesis, chorionic villi sampling (CVS), and regular scheduled ultrasound allow parents to monitor the health of the growing fetus.

Newborn Screening• The most widespread type of genetic screening,

newborn screening is used to detect genetic or metabolic conditions for which early diagnosis and treatment are available.

• State tests for newborns typically screen anywhere from 4 to over 30 genetic or metabolic disorders. – Testing protocol and mandates vary from state to state.

• The goal of newborn screening is to identify affected newborns quickly in order to provide quick treatment and care.

2.1.3Test Genes (PTC bitter taste)

PTC ActivityWhile synthesizing a chemical called phenylthiocarbamide (PTC) in his lab, scientist Arthur Fox accidentally released some into the air.

Fox’s colleague in the lab complained that the dust had a very bitter taste. Fox, however, tasted nothing.

After further studies, scientists concluded that the inability to taste PTC is actually a recessive trait. Bitter-tasting compounds are recognized by receptor proteins on the surface of taste cells. The gene for this PTC taste receptor, TAS2R38, was identified in 2003. Sequencing identified three variations in this gene from person to person. These base pair differences, or SNPs, correlate to a person’s ability to taste PTC.

What are SNPs? How can restriction enzymes and electrophoresis be used to identify SNPs

and determine genotype?

SNP = Single Nucleotide Polymorphism – A single base pair change.

Step 1: Isolating DNA

• The gene of interest in the experiment, TAS2R38, is located on chromosome #7. This gene is associated with our ability to taste a chemical called PTC.

• In this lab you isolated a DNA sample from your cheek cells.

Step 2: Amplifying the Gene of Interest

• Using your DNA sample, you amplified a 220 base pair region of the PTC gene using PCR.– Specific primers attach to either side of the target sequence

• You investigated one of the base pair changes or single nucleotide polymorphisms (SNPs) that affects a person’s ability to taste the chemical PTC.

What is the goal of PCR? What are the steps of the PCR process?

What is the relationship between phenotype and genotype?

• Genotype - Homozygous Dominant – RR – Have two copies of the taster gene = Phenotype – Strong Taster

• Genotype – Heterozygous – Rr – Have one copy of the taster gene = Phenotype – Weak taster

• Genotype – Homozygous Recessive – rr – Have two copies of the non-taster gene = Phenotype – No taste

Genetics Review – Question 1

• The inability to taste PTC is a recessive trait. • If a capital “T” is is used to designate the dominant

allele and a lowercase “t” is used to designate the recessive allele, what is the genotype of a “Nontaster”?

Answer

A “Nontaster” carries two recessive alleles and thus has the genotype “tt”

Genetics Review – Question 2

What are the possible genotypes for a “Taster”?

Answer

A “Taster” may be homozygous dominant with a genotype of “TT” or heterozygous with a genotype of “Tt”.

Step 3: Restriction Analysis

• Restriction enzymes, molecular scissors, recognize specific DNA sequences and cut the nucleotide strands.

• In this part of the experiment, you will use a specific restriction enzyme, HaeIII, to identify a SNP or base pair difference in the amplified segment of the PTC tasting gene.

Step 4: Gel Electrophoresis

• Gel Electrophoresis separates DNA fragments based on their molecular weight.

• Once you have digested your DNA sample with the restriction enzymes, run your product on a gel to analyze your results.

PCR

The DNA, DNA polymerase, buffer, nucleoside triphosphates, and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler

PCR Thermocycler

The three main steps of PCR• The basis of PCR is temperature changes and the effect that these

temperature changes have on the DNA. • In a PCR reaction, the following series of steps is repeated 20-40 x (note: 25 cycles usually takes about 2 hours and amplifies the DNA

fragment of interest 100,000 fold)Step 1: Denature DNA At 95C, the DNA is denatured (i.e. the two strands are separated)

Step 2: Primers Anneal At 40C- 65C, the primers anneal (or bind to) their complementary

sequences on the single strands of DNA

Step 3: DNA polymerase Extends the DNA chain At 72C, DNA Polymerase extends the DNA chain by adding

nucleotides to the 3’ ends of the primers.

Step 1:DenaturationdsDNA to ssDNA

Step 2:AnnealingPrimers onto template

Step 3:ExtensiondNTPs extend 2nd strand

Step 1:DenaturationdsDNA to ssDNA

Step 2:AnnealingPrimers onto template

Step 3:ExtensiondNTPs extend 2nd strand

Vierstraete 1999

extension products in one cycle serve as template in the nextextension products in one cycle serve as template in the next

Heat-stable DNA Polymerase

• Given that PCR involves very high temperatures, it is imperative that a heat-stable DNA polymerase be used in the reaction.

• Most DNA polymerases would denature (and thus not function properly) at the high temperatures of PCR.

• Taq DNA polymerase was purified from the hot springs bacterium Thermus aquaticus in 1976

• Taq has maximal enzymatic activity at 75 C to 80 C, and substantially reduced activities at lower temperatures.

2.1.5Fetal Health

Amniocentesis versus CVS

Chorionic Villuschorion is one of the four extraembryonic

membranes that make up the amniotic egg

2.2.1Gene Therapy