Cytogenetics
Area of genetics that links chromosome variations to specific traits including illnesses.
Monosomic vs. Trisomic Conditions
-Monosomic Conditions are lethal
-There are only 3 trisomic diseases
What are 3 essential parts of a chromosome?
-What is the difference between heterochromatin and euchromatin and which is more active?
-Telomeres- Necessity for linear DNA. Telomerase (often found in cancer) synthesizes more ends of the chromosome and has its own primer. They are composed of many repeats of TTAGGG and shorten with each cell division.
-Origins of replication sites
-Centro
Centromeres
-Bases that form centromeres are repeats of a blank base DNA sequence
-Replicated at end of what and facilitated by what protein?
-CENP-A is an example of what kind of change?
The largest constriction of the chromosome and where spindle fibers attach
The bases that form the centromere are repeats of a 171-base DNA sequence. These sequences have mainly a structural role instead of an informational role.
Replicated at the end of
Subtelomeres
-Between what and what
-Consists of how many bases?
-How many protein encoding genes?
The chromosome region between the centromere and telomeres
Consists of 8,000 to 300,000 bases
Near telomere the repeats are similar to the telomere sequence
Contains at least 500 protein-encoding genes
- About 50% are multigene families that include pseud
-Telocentric
-Acrocentric
-Submetacentric
-Metacentric
-Which one of these do humans not have?
-A Chromosome is metacentric if the centromere divides it into two arms of approximately equal length.
-It is Submetacentric if the centromere establishes one long arm and one short arm
- It is acrocentric if it pinches off only a small amount of material
DNA Probe
Labeled piece of DNA tat binds to its complementary base sequence on a particular chromosome.
Chromosome microarray analysis
Can be paired with the older techniques of a chromosome study to detect copy number variants CNV, which include extremely small sections of missing or extra DNA.
Chorionic Villi Sampling
Performed when in the pregnancy?
-Does not detect what?
-How are these cells identical to the fetus?
Performed during 10-12th week of pregnancy.
-Obtains cells from the chorionic villi, which are fingerlike structures that develop into the placenta.
Provides earlier results than amniocentesis However, it does not detect metabolic problems because it does
-AFP
-hCG
Alpha Fetoprotein
Human Chorionic Gonadotropin
Fetal Cell Sorting
-How are fetal cells distinguished from maternal cells?
-A new technique detects fetal blank in the bloodstream of the mother.
Fetal cells are distinguished from maternal cells by a fluorescence-activated cell sorter - Identifies cell-surface markers
A new technique detects fetal mRNA in the bloodstream of the mother.
How were staining techniques improved to allow more chromosome bands to be seen?
In the 1970s, improved staining techniques gave banding patterns unique to each chromosome
Then researchers found that synchronizing the cell cycle of cultured cells revealed even more bands per chromosome
FISH
Florescence in situ hybridization
DNA probes labeled with fluorescing dye bind complementary DNA.
FISH is much more precise than conventional chromosome staining because it specifically attaches to some DNA sequences and illuminates them.
45, X
47, XYY
46, XY, del (7q)
46,XY, t(7;9)(p21.1; q34.1)
45, X- Turner syndrome
47, XYY- Jacobs Syndrome
46, XY, del (7q)- Male missing part of the long arm of chromosome 7
46,XY, t(7;9)(p21.1; q34.1)- A male with a translocation between the short arm of chromosome 7 at band 21.1 and the long arm of chromosome
Ideogram
A schematic chromosome map
Indicates chromosome arms (p or q) and delineates major regions and subregions by numbers.
Abnormal chromosomes account for at least blank% of spontaneous abortions.
A karyotype may be abnormal in two ways: 1) In chromosome number
2) In chromosome structure
Abnormal chromosomes account for at least 50% of spontaneous abortions yet only .65% of newborns have them.
Due to improved technology, more people are being diagn
Polyploidy - definition
-How many sets of chromosomes do triploids have?
-how is it produced?
-Account for blank% of spontaneous abortions and blank% of stillbirths.
Cell with extra chromosome sets is polyploid Triploid (3N) cells have three sets of
chromosomes
- Produced in one of two main ways:
- Fertilization of one egg by two sperm
- Fusion of haploid and diploid gametes
Triploids account for 17% of all spontaneou
Aneuploidy
-A normal chromosomal number is called a blank.
-Cells with extra or missing chromosomes are blank.
-Do a lot survive? if they do what those that are born are more likely to have blank compared to blank.
A normal chromosomal number is euploid Cells with extra or missing chromosomes
are aneuploid
Most autosomal aneuploids are
spontaneously aborted
Those that are born are more likely to have an extra chromosome (trisomy) rather than a missing one (monosomy)
Nondisjunction
-Failure of what during what?
Nondisjunction during Meiosis I results in what whereas nondisjunction during Meiosis II results in what?
The failure of chromosomes to separate normally during meiosis
Produces gamete with an extra chromosome and another with one missing chromosome
Nondisjunction during Meiosis I results in copies of both homologs in one gamete
Nondisjunction during Meiosis
What are the most frequently seen trisomies in newborns?
Most autosomal aneuploids cease developing as embryos or fetuses
Most frequently seen trisomies in newborns are those of chromosomes 21 (Down) 85% survive, 18 (Edward) 5% survive, and 13 (Patau) 5% survive.
- Carry fewer genes than other autosomes
Aneuploidy and polyploidy also arise during what?
-An individual with two chromosomal distinct cell populations is a what?
Aneuploidy can also arise during mitosis, producing groups of somatic cells with the extra or missing chromosomes
An individual with two chromosomally-distinct cell populations is called a mosaic
A mitotic nondisjunction event that occurs early in develop
Autosomal Aneuploids
Most autosomal aneuploids cease developing long before birth. The most frequently seen extra autosomes in newborns are chromosomes 21, 18, and 13 because these chromosomes carry many fewer protein-encoding genes than the other autosomes which allow the fe
Down Syndrome
-Originally termed blank
-Individuals are more likely to develop what?
-Why are they more likely to get Alzheimer disease?
Down syndrome
Most common trisomy among newborns
Distinctive facial and physical problems
Figure 13.13
Varying degrees of developmental disabilities Individuals more likely to develop leukemia
Link with one form of Alzheimer disease because these patients
3 Reasons why as a woman ages, she is more likely to have a child with Down Syndrome.
1) Long time her oocytes have been arrested on the brink of completing meiosis. Oocytes have been exposed to toxins, viruses, and radiation during this time.
2)Females have a pool of immature aneuploid oocytes resulting from spindle abnormalities, as a wo
Edwards Syndrome
-Most due to nondisjunction in blank in oocyte
-What are some symptoms and abnormalities?
Most due to nondisjunction in meiosis II in oocyte and do not survive
Serious mental and physical disabilities
-A distinctive feature: Oddly-clenched fists, heart defects, a displaced liver and growth retardation.
Patau Syndrome
-Trisomy blank.
-What is a distinct feature?
-Very rare and generally do not survive 6 months
-Serious mental and physical disabilities A distinctive feature: Eye fusion, also could have major abnormalities affecting the heart, kidneys, brain, face, and limbs.
Turner Syndrome
1 in blank female births
How many of these fetuses die in utero?
-Possible features include
-Individuals who are blank may have children.
Called the XO syndrome
1 in 2,500 female births
99% of affected fetuses die in utero
Features include short stature, webbing at back of neck, incomplete sexual development (infertile), impaired hearing
Individuals who are mosaics may have children.
Triplo-X
1 in blank female births
What are the modest effects on phenotype?
-What is special about the type of X-inactivation that occurs?
-Called the XXX syndrome
-1 in 1,000 female births
Few modest effects on phenotype include tallness, menstrual irregularities, and slight impact on intelligence
X-inactivation of two X chromosomes occurs and cells have two Barr bodies
May compensate for p
Klinefelter Syndrome
1 in blank male births
-What are some of the phenotypes
Called the XXY syndrome 1 in 500 male births Phenotypes include:
- Incomplete sexual development
-Rudimentary testes and prostate
-Long limbs, large hands and feet
-Some breast tissue development
-Most common cause of male infertility.
XXYY Syndrome
-Arises due to unusual blank and blank.
-More severe blank problems than Klinefelter syndrome.
-Individuals are blank.
-Treated with blank.
-Likely arises due to unusual oocyte and sperm.
-Associated with more severe behavioral problems than Klinefelter syndrome.
- AAD, obsessive compulsive disorder, learning disabilities
-Individuals are infertile Treated with testosterone.
XYY Syndrome
-Also known as what?
-1 in blank male births
-Modest phenotypes include what?
-Studies suggesting increase in blank behaviors are not supported.
Why is YO impossible?
Also known as Jacobs syndrome 1 in 1,000 male births
96% are phenotypically normal
Modest phenotypes may include great height, acne, speech and reading disabilities
Studies suggesting increase in aggressive behaviors are not supported
May arise from nondi
Deletions and Duplications
-Are they inherited?
-What does de novo mean?
-Cri-du-chat (cat cry) syndrome is a deletion on blank p. Why is it called cat cry syndrome?
A deletion refers to a missing genetic segment from a chromosome
Deletions are often not inherited
-Rather they arise de novo
Larger deletions increase the likelihood that there will be an associated phenotype
Cri-du-chat (cat cry) syndrome
- Deletion 5p
Duplication
-Refers to the presence of what on a chromosome
-are they inherited
-Which is worse, a larger or smaller duplication?
A duplication refers to the presence of an extra genetic segment on a chromosome
Duplications are often not inherited - Rather they arise de novo
The effect of duplications on the phenotype is generally dependent on their size
- Larger duplications tend t
Robertsonian translocations
-Where do chromosomes break and how many chromosomes do translocation carriers have?
Two nonhomologous acrocentric chromosomes break at the centromere and their long arms fuse
- The short arms are often lost causing the large arms to join and form a single large chromosome with 2 long arms.
Affect 1 in 1,000 people
Translocation carriers
Translocation Down Syndrome
About blank% of Down Syndrome results from a blank translocation between chromosomes blank and blank.
-Does it recur in families?
About 5% of Down syndrome results from a Robertsonian translocation between chromosomes 21 and 14
Tends to recur in families, which also have more risk of spontaneous abortions
One of the parents is a translocation carrier
- They may have no symptoms
- Ho
Reciprocal Translocation
-How do two nonhomologus chromosomes interact?
-Normally healthy, but what could occur to cause a phenotype?
Two nonhomologous chromosomes exchange parts
About 1 in 500 people are carriers
- Are usually healthy because they have the normal amount of genetic material (but it is rearranged)
However, if the translocation breakpoint interrupts a gene, there may be a
Inversion
-A chromosome segment that is blank in orientation.
-Paracentric vs. pericentric inversion
-An inversion is a chromosome segment that is flipped in orientation
-5-10% cause health problems probably due to disruption of genes at the breakpoints
-Paracentric inversion = Inverted region does NOT include centromere
-Pericentric inversion = Inverted
Isochromosomes
-Chromosomes with blank arms.
-Form when blank divide along the incorrect plane during meiosis.
-How does the splitting of the chromatids at the centromere differ in isochromosomes?
-Chromosomes with identical arms
-Known for chromosomes 12 and 21 and for the long arms of X and the Y.
-Form when centromeres divide along the incorrect plane during meiosis.
-Instead of splitting sagitally, it splits in a transverse manner.
Ring Chromosomes
-When might they arise?
-Genes can be blank or blank causing symptoms.
-Exposure to blank forms rings.
-Dicentric and acentric chromosomal abnormalities.
-Ring Chromosomes
-Occur in 1 in 25,000 conceptions
May arise when telomeres are lost and sticky chromosome ends fuse
Genes can be lost or disrupted causing symptoms.
Dicentric and a centric is a crossover between a chromosome with a paracentric inversion
UPD
Inheritance of blank chromosomes or chromosome parts from the blank parent
-Requires the simultaneous occurrence of two rare events that are separate.
Inheritance of two chromosomes or chromosome parts from the same parent
UPD requires the simultaneous occurrence of two rare events
1) Nondisjunction of the same chromosome in both sperm and egg
OR
2) Trisomy followed by chromosome loss
-This may cause Pr
Chapter 18
...
Basics of cancer
-Dedifferentiate cells in the body.
-p53 gene may not be functional in a lot of cancers.
-Uncontrolled cell growth.
-Can be either acceleratory or inhibitory.
Benign vs. Cancerous
-Cancer cells don't usually make blank and blank
-What is metastasis?
-A tumor is benign if it grows in place but does not spread into, or invade surrounding disuse.
-A malignant tumor infiltrates nearby tissues and also sends parts of itself into the bloodstream or lymphatic vessels.
-Cancer cells don't usually make CAM's
Melanocytes
Reside in the basal layer of cells. If you make a lot of melanin, your dark. When melanin gets dark, it makes the "umbrella" darker and therefore shades the basal layer of the skin.
-Cancer Causing Genes
-Only about blank% of genes are inherited as blank gene disorders in which the faulty instructions are in blank cell.
-Oncogenes-how many and when do they cause cancer?
-Tumor suppressor genes- how many and when do they cause cancer?
-Only 10% of cases are inherited as a single-gene disorder, in which the faulty instructions are in every cell.
-Oncogenes
- More than 100
- Cause cancer if inappropriately activated. Cancer begins in a single cell when an oncogene is turned on or a tumor
Cell Cycle Control
-What do timing, rate, and number of cell divisions depend on?
-p53 issues.
Timing, rate, and number of cell divisions depend on:
- Protein growth factors
- Signaling molecules from outside the cell - Transcription factors within
Checkpoints control the cell cycle
- Ensure that mitotic events occur in the correct sequence.
-If p5
Loss of cell cycle control
-Many types of cancer result from what type of check points
-When is the S phase checkpoint?
-When is the apoptosis checkpoint in the cell cycle?
Many types of cancer result from faulty check points
Cancer sends a cell down a pathway of unrestricted cell division
Cancer cells either lose specializations or never specialize
-The DNA damage checkpoint is in S phase.
-Apoptosis checkpoint is after G2
Importance of telomeres and telomerase
-Normal vs. cancer cells in action of telomerase.
Loss of control of telomere length may also contribute to cancer
Telomerase is the enzyme (complex of RNA and protein) that adds telomere sequences to the ends of chromosomes
Normal, specialized cells have telomerase turned off, limits cell division
Cance
Inherited vs. Sporadic Cancer
-Somatic mutations- inheritable?
-Germline mutations.
-In a germline cancer, every blank has one gene variant that increases cancer blank, and a second mutation in a cell of the blank tissue.
Examples.
Somatic mutations
- Occur sporadically in nonsex cells
- Result from a single dominant mutation or two recessive mutations in the same gene
- Cancer susceptibility not passed on to offspring
Germline mutations
-In a germline cancer, every cell has one gen
Origin of Cancer
-Begins at the blank and blank levels
Cancer begins at the genetic and cellular levels
If not halted, cancer spreads through tissues to take over organs and organ systems
The origin and spread of cancer are summarized next
Characteristics of Cancer cells
-Divide blank
-Contain what
-Cell surface has a different what?
-Many cancer cells are blank (referring to # of chromosomes)
Divide continually (given space and nutrients), and quicker than normal cells
Contain heritable mutations
Transplantable
Dedifferentiated: lose their specialized identity
Have a different appearance
Cell surface has different types and/or number of antige
Cancer cells lack contact blank
-They induce blank
-They are invasive how?
-Lack contact inhibition
-Induce angiogenesis: formation of local blood vessels
-Invasive: squeeze into any space available. They also anchor themselves to tissue membranes and secrete enzymes that cuts paths through healthy tissues. They also break down
VEGF
Vascular Endothelial Growth Factor
-stimulates nearby capillaries to sprout new branches that extend toward the tumor, bringing in oxygen and demoing wastes. This is called Angiogenesis
What are the four ways in which cancer can begin at a cellular level?
Cancer can begin at the cellular level in at least four ways:
- Activation of stem cells that produce cancer cells
- Dedifferentiation
- Increase in proportion of a tissue that consists of stem cells or progenitor cells
- Faulty tissue repair
IPS
Induced Pluripotent Stem cells
-How can cancers from shifting the balance of cell types in a tissue arise?
-Uncontrolled tissue repair can cause tissue how?
-If mutation renders a differentiated cell to divide to yield other cells that frequently divide, then over time these cells may take over, forming an abnormal growth.
-If epithelium is occasionally damaged, resting stem cells can become activate and divi
What are proto-oncogenes?
-What happens when proto-oncogenes are turned on at the wrong time?
-What are oncogenes?
Proto-oncogenes are normal versions of genes that promote cell division normally found at a wound site.
Expression at the wrong time or in the wrong cell type leads to cell division and cancer
Proto-oncogenes are called oncogenes in their mutated form
One
Blank integrated next to a pro to-oncogene can cause what.
-Moving a proto-oncogene next to a highly transcribed gene can lead to what
-An example of this is what?
Viruses integrated next to a proto-oncogene can cause transcription when the virus is transcribed
Moving a proto-oncogene next to a highly transcribed gene can lead to overexpression of the proto-oncogene
Example: Burkitt lymphoma
- A translocation places
Burkitt Lymphoma
People are infected with the Epstein-Barr virus, which stimulates specific chromosome movements in maturing B cells to assemble antibodies against the virus.
-Caused by a translocation between chromosome 8 and 14.
Fusion Proteins
-What gets activated when a pro to-oncogene moves next to another gene?
-The gene pair is blank together
-The double gene product is a blank protein.
-What do fusion proteins do?
Oncogenes are activated when a proto- oncogene moves next to another gene
The gene pair is transcribed together
The double gene product is a fusion protein - It activates or lifts control of cell division.
Acute Promyelocytic Leukemia
-Translocation where?
-Combination of blank acid cell surface receptor and what?
-What does the fusion protein function as?
-Some patients respond to what type of drugs.
-Translocation between chromosomes 15 and 17
Combination of retinoic acid cell surface receptor and an oncogene, myl
Fusion protein functions as a transcription factor
- When overexpressed causes cancer Some patients respond to retinoid drugs
CML
-What is the chromosome connected with it and what type of chromosomal abnormality comes along with it?
-What drug is helping to treat it?
Chronic Myelogenous Leukemia
-Arises from the "Philadelphia chromosome" which is a result of a translocation between chromosome 9 one gene (alb Abelson oncogene) and another gene from 22 (bcd breakpoint cluster region) fuse together to form the encoded fu
Her-2/neu
-In about 25% of women with what?
-Affected cells have blank to blank million copies of HER2 which is the product of a blank.
-In HER2 breast cancer, too many receptors send too many what to divide.
Product of an oncogene
Excessive levels in approximately 25% of breast cancer patients
-Affected cells have 1 to 2 million copies of HER2 which is a product of an oncogene.
Too many receptors
Too many signals to divide
Monoclonal antibody drug, Herceptin,
Tumor Suppressor
-Another way that cancer can be caused by loss of genes that inhibit blank.
-Mutations in blank copies of a tumor suppressor gene is usually required to allow cell division.
-Blank is another way genes can be lost.
-Cancer can be caused by loss of genes that inhibit cell division
-Tumor suppressor genes normally stop a cell from dividing
-Mutations of both copies of a tumor suppressor gene is usually required to allow cell division
-Genes can also be lost by deletio
RB
The Retinoblastoma gene is on what chromosome and on what arm?
-RB protein normally binds what so that they cannot blank genes that carry out mitosis.
-What is the "two-hit" hypothesis? Is it required to cause a cancer related to tumor suppressor delet
A rare childhood cancer
The RB gene is on chromosome 13 on the long arm.
The RB protein binds transcription factors so that they cannot activate genes that carry out mitosis
- Normally halts the cell cycle at G1
Study of RB was the origin of the "two-hit
Two hit Hypothesis
-How does it differ between sporadic cases and familial cases?
Two mutations or deletions are required - One in each copy of the RB gene
For sporadic cases (non-inherited)
- Retinoblastoma is a result of two somatic mutations
For familial cases (inherited)
- Individuals harbor one germline mutant allele
for the RB ge
p53
-Either repairs or can induce blank
-More than blank% of humor cancers involve an abnormal what?
-Rare inherited mutations in the p53 gene cause what disease?
-The p53 gene is the "guardian of the genome"
-Determines if a cell has repaired DNA damage
-If damage cannot be repaired, p53 can induce apoptosis
-More than 50% of human cancers involve an abnormal p53 gene
-Rare inherited mutations in the p53 gene caus
Breast Cancer can arise in two forms, what are they and how many somatic mutation need to occur for each?
Two main forms
- Familial form: A germline mutation is inherited and then a somatic mutation occurs in a breast cell
- Sporadic form: Two somatic mutations affect the same cell
Mutations in many genes can cause cancer
BRCA1 and BRCA2 -What do they stand for?
-Encode proteins that join two others to form a complex for what function?
-Breast Cancer Predisposition Gene 1 or 2"
-The two major breast-cancer susceptibility genes are BRCA1 and BRCA2
- Encode proteins that join two others to form a complex that allows repair of double-stranded DNA breaks
-Mutations in these genes have diffe
ATM and CHEK2 genes can also cause breast cancer. -How?
ATM gene product adds what to CHEK2 product, which then does what?
-Genes whose protein products affect those of BRCA1, BRCA2, and p53 can cause breast cancer
-Example: The ATM gene product adds a phosphate to the CHEK2 gene product, which then adds a phosphate to the BRCA1 protein
- Mutations in ATM and CHEK2 can cause
MicroRNA mutations
-MicroRNA's normally control the expression of blank and blank.
-What could this result in?
MicroRNAs normally control the expression of proto-oncogenes and tumor suppressor genes
- Thus, when they are mutated or differentially expressed, cancer can result
Patterns of microRNA expression change as a cancer progresses
- This is being used to deve
Gatekeeper vs. Caretaker genes
Gatekeeper genes
- Directly control mitosis and apoptosis
Caretaker genes
- Control mutation rates and may have an overall effect, when mutant, in destabilizing the genome
Most cancers are the culmination of a series of mutations in several genes
FAP
-Blank genes mutate
-Activation of blank
-Mutations in blank, blank, and other genes
-Blank triggers metastasis
-Blank genes cause genomic instability.
FAP- Familial Adenomatous Polyposis
5% of colon cancer cases are inherited 1 in 5000 in U.S. has FAP
Causes multiple polyps at an early age Several mutations contribute
- APC genes mutate
- Activation of oncogenes (E.g. K-Ras)
- Mutations in TGF, p53, and
The Cancer Genome
-What are the functions of atlases?
Several large-scale projects are analyzing genomes of cancer cells
- These allow construction of descriptive "atlases" containing different types of information
Many mutations accompany cancer, but they interact in only a few pathways. Purpose is to ident
How do environmental factors contribute to cancer?
-Inheriting a susceptibility gene places a person farther along the road to blank.
Environmental factors contribute to cancer by mutating or altering the expression of genes that control the cell cycle, apoptosis, and DNA repair
Inheriting a susceptibility gene places a person farther along the road to cancer
- However, cancer can happe
Population Study vs. Case Control Study vs. Prospective Studies
-Population Study: Compares incidence of a type of cancer among different groups of people
-Case Control: Identify differences between patients with a type of cancer and healthy individuals matched for multiple characteristics
-Prospective Studies: Two or
ALL
MLL
Acute Lymphoblastic Leukemia
Mixed-Linkage Leukemia
DNA Microarrays
Scan the genome for cancer-associated mutations as well as gene expression patterns.
New Types of Cancer Diagnosis and Treatment
New types of cancer drugs:
- Stimulate cells to regain specialized characteristics
- Inhibit telomerase
- Induce apoptosis
- Inhibit angiogenesis
Genomics information is increasingly used
- Enables physicians to better match patient to treatment.
Biotechnology definition
-What is a transgenic organism
-Recombinant DNA comes from what
-How are both of these phenomenons possible?
Biotechnology is the use or alteration of cells or biological molecules for specific applications
A transgenic organism has DNA from different species
Recombinant DNA comes from more than one type of organism
Both are possible because of the universality
PCR
-Where does it work on DNA?
-How many times does it replicate sequence?
Recombinant DNA Technology Amplifies DNA within cells often using what from other sequences
(PCR) Polymerase Chain Reaction-- Works on DNA molecules outside of cells
- Replicates sequence millions of times
-PCR is very common in forensics/DNA printing
-Done in vitro, needs enough sequence info to generate primers.
Recombinant DNA Technology- "cu
What are the 3 basic steps in PCR and what are they used for?
-What is the enzyme used in PCR and why is it this specific enzyme?
-What is PCR's biggest strength and biggest weakness?
For PCR you don't have to purify anything. Need to be careful that you don't contaminate it with your own DNA. It uses DNA polymerase to rapidly replicate a specific sequence of DNA.
Consists of a repetition of three basic steps:
1. Denaturation: Heat is
Transcription-Mediated Amplification
Copies target DNA into blank and then uses blank to amplify RNA.
-Does not require blank
-Generates how many copies per cycle?
-Can yield how many copies of a selected sequence in 30 minutes
Copies target DNA into RNA and then uses RNA polymerase to amplify RNA
Does not require temperature shifts
Generates 100 to 1,000 copies per cycle, compared to PCR's doubling
- Can yield 10 billion copies of a selected sequence in 30 minutes
Recombinant DNA Technology
-Also known as blank cloning
-Steps to create recombinant DNA.
-Cut, Mix, and mass produced, how do they do this?
-What cuts the DNA?
-What carries the DNA? What are examples of cloning vectors?
-How does it replicate?
-Recombinant DNA technology is also known as gene cloning
-They first cut the donor and recipient DNA with restriction enzymes.
-Then, these DNA fragments are carried by the cloning vectors such as Plasmids, bacteriophages, and disabled retroviruses.
-Whe
Genomic Library
All the DNA in donor cell gets cut and includes introns and exons. Researchers assemble collections of recombinant bacteria that harbor pieces of a genome, the pieces overlap where the sequences align. This is called a genomic library.
-To find a specific
-Genomic library
-DNA Probe
-cDNA library
-Genomic library: Collections of recombinant DNA that contain pieces of the genome.
-DNA probe: Radioactively (or fluorescently) labeled gene fragments.
-cDNA library: Genomic library of protein encoding genes produced by extracting mRNA and using reverse
Importance of cDNA
If you use want to see the DNA that codes for proteins, you just need to find a mRNA transcript from a differentiated cell, use reverse transcriptase to, turn it back into DNA, and then you have the DNA of only protein coding DNA.
Vectors are commonly engineered to carry what
Vectors are commonly engineered to carry antibiotic resistance genes
Host bacteria without a plasmid die in the presence of the antibiotic
Bacteria harboring the vector survive
Growing cells on media with antibiotics ensures that all growing cells must ca
Selecting for cells with inserted DNA
-How color plays a role
The site of insertion of the DNA of interest can be within a color-producing gene on the vector
Insertion of a DNA fragment will disrupt the vector gene
- And so the bacterial colony that grows will be colorless.
Applications of Recombinant DNA
-Study the biochemical properties or genetic pathways of what
-Mass produce what?
-How can the textile industry produce indigo dye in E.coli?
Recombinant DNA is used to:
- Study the biochemical properties or genetic pathways of that protein
- Mass-produce proteins (e.g., insulin)
Sometimes conventional methods are still the better choice because of economics
Textile industry can produce indigo
What kind go recombinant genes do sheep, cows, and goats have in their milk?
An even more efficient way to express some recombinant genes is in a body fluid of a transgenic animal
Transgenic sheep, cows, and goats have all expressed human genes in their milk,
- Clotting factors - Clot busters
- Collagen
- Antibodies
What are some techniques that are used to insert DNA into cells to create transgenic animals?
Several techniques are used to insert DNA into cells to create transgenic animals
- Chemicals that open transient holes in plasma membrane
- Liposomes that carry DNA into cells
- Electroporation: A brief jolt of electricity to open membrane
- Microinjecti
What is the importance of animal models in recombinant cells?
Transgenic animals are far more useful as models of human diseases
- Example: Inserting the mutant human beta globin gene that causes sickle-cell anemia into mice
Drug candidates can be tested on these animal models before testing on humans
- Will be aban
Limitations of transgenic animal models (3).
Transgenic animal models have limitations
- Researchers cannot control where a transgene inserts, and how many copies do so
- The level of gene expression necessary for a phenotype may differ in the model and humans
- Animal models may not mimic the human
Bioremediation
Transgenic organisms can provide processes as well as products
Bioremediation: The use of bacteria or plants to detoxify environmental pollutants
Examples
- Nickel-contaminated soils
- Mercury-tainted soils
- Trinitrotoluene (TNT) in land mines
Gene Expression DNA Microarrays
-display what?
Gene expression DNA microarrays
(gene chips) are devices that detect and display the mRNAs in a cell
A microarray is a piece of glass or plastic that is about 1.5 centimeters square
Many small pieces of DNA of known sequence are attached to one surface, i
How it works
mRNAs are extracted and cDNAs are made. The cDNA's from the injury are dyed red, the cDNA's from the control are dyed green.
A laser scanner than detects and converts the results to a colored image. It can bind DNA pieces from both samples, either, or nei
What are 3 techniques that can be used to control gene expression?
-When could silencing DNA be useful?
In some situations, silencing gene expression may be useful
- Blocking transcription of oncogenes
Three techniques can be used to control gene expression
- RNA interference
- Antisense sequences
- Knockouts from gene targeting
Single stranded vs. RNA interference
Single-stranded RNAs can fold into short, double-stranded regions (hairpins) when the sequence is complementary.
Short, double-stranded RNAs sent into cells separate into single strands
- One of these strands binds its complement in mRNA, preventing it fr
Antisense-induced Exon Skipping
-Silences mutations that causes what
-Morpholinos- bind to their complement allowing for what?
Antisense-induced exon skipping silences mutations that cause exons to be cut out of maturing mRNA
Researchers introduce short, synthetic DNA molecules called morpholinos
- Complementary or "antisense" to parts of the gene with the splicing mutation
Morph
Gene targeting
-Replaces a normal DNA sequence with what?
Gene targeting is a technique that uses homologous recombination to replace a normal DNA sequence with one that cannot be transcribed or translated
- This silences gene expression by creating a "knockout" gene
- Moreover, observing what happens (or not) c
Pharmacogenetic Test vs. a Pharmacogenomic test
A pharmacogenetic test detects a variant of a single gene that affects drug metabolism
A pharmacogenomic test detects variants of multiple genes or gene expression patterns that affect drug metabolism
These are both known as "personalized medicine."
Selec
Gene Therapy
-Theoretically can provide a longer-lasting effect compared to treating what?
-Now its targeting more common issues such as what?
Altering genes theoretically can provide a longer-lasting effect than treating symptoms
The first efforts focused on inherited disorders with a known mechanism, even though the conditions are rare
Gene therapy now is targeting more common illnesses, such
Germline vs. Somatic gene therapy
Germline gene therapy
- Gamete or zygote alteration; heritable; not done in humans; creates transgenic organisms. Alters the DNA of a gamete or fertilized ovum, as a result, all of the cells of the individual have the change. The change is hereditary and
-Ex vivo gene therapy.
-In situ gene therapy
-In vivo gene therapy
-Which is the most invasive?
Ex vivo gene therapy is applied to cells outside of body that are then returned. Remove cells, alter them, and return them to the bloodstream.
In situ gene therapy occurs directly on accessible body parts
In vivo gene therapy is applied directly to an int
Gene Delivery
What are the 3 methods and what are some examples of them?
Physical methods
- Electroporation, microinjection, and particle bombardment
Chemical methods
- Liposomes and other types of lipids
Biological approaches
- A vector (modified viral genomes). Researchers remove the viral genes that cause symptoms or alert
AAV
ADA
-Adenoassociated virus
-Adenosine Deaminase Deficiency
Ashanti Illness and treatment
Ashanti has an inherited immune deficiency due to adenosine deaminase deficiency.
-She then received gene therapy when they took out her cells, corrected the WBC's and put them back in her.
Jesse Gelsinger
Had OTC
Jesse Gelsinger had ornithine transcarbamylase deficiency in which one of five enzymes made in the liver is absent. Instead of being excreted in the urine, the ammonia accumulates in the bloodstream.
-Was given an adenovirus carrying a functional human OT
SCID-X1
-Importance of a retrovirus in gene therapy
X-linked Severe Combined Immune Deficiency. T cells lack certain cytokine receptors preventing the immune system from recognizing infection.
-From Jesse's situation, the doctors realized that they could not use adenovirus's anymore and therefore used retr
Canavan disease affects the brain.
-Why is it a good gene therapy candidate (5)?
Causes brain degeneration in children
Good gene therapy candidate because: 1. Gene and protein are well known 2. Window of time exists for treatment 3. Only the brain is affected
4. Brain scans can be used to monitor treatment
5. No existing treatment
Lack of what enzyme disrupts what?
-Neurons release what
-What is normally broken down by the enzyme blank
-What happens with an enzyme deficiency?
-Lack of oligodendrocytes prevents what?
An aspartoacylase enzyme deficiency
Neurons release N-acetylaspartate (NAA)
NAA is normally broken down by the enzyme aspartoacylase to harmless components
Enzyme deficiency creates NAA buildup, which destroys oligodendrocytes
Lack of oligodendrocytes pre
Leber's Congenital Amaurosis II
-What is it
-What are these individuals missing?
A layer called the retinal pigment epithelium cannot make an enzyme necessary to convert vitamin A to a form that the rods and cones can use.
-Adeno-associated virus