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GN 311 > Test 5 > Flashcards

Test 5 Flashcards

1
Q

Evolution

A

GENETIC changes in the composition of a population including:

  • emergence of species
  • divergence of species
  • extinction of species

Involves variation, heredity, and selection:
If variation is not heritable, then it cannot be passed to progeny

-Study diversity that exists in a population and between populations and the factors that can cause diversity.

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2
Q

Selection

A

works on entire organisms phenotype so many loci as well as environmental factors are important

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3
Q

Genetic change in populations

A

2 step process:

Changes occurs (must be genetic)

  • eg. mutation causes new alleles
  • eg. recombination causes new combinations of alleles

Then different alleles (or combinations) must increase or decrease in frequency in the gene pool (selection and other factors)

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4
Q

What are some factors effecting diversity between populations

A 
Genetic Drift
Migration
Mutations
Selection
Inbreeding
Natural Selection
Recombination
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5
Q

Genetic drift

A

results in divergence as some populations become fixed for each allelic form

-decreases diversity within populations as alleles are fixed and lost

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6
Q

Migration

A

tends to equalize population

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7
Q

Selection

A

can cause divergence between populations if different alleles are favored in different populations

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8
Q

What does different mutations in different populations allow?

A

populations to diverge

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9
Q

Migration and mutation

A

introduce variability within populations by introducing new alleles

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10
Q

Inbreeding

A

increases homozygous types with decrease of heterozygous types

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11
Q

Natural selection

A

can increase or decrease variability within population depending on type of selection

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12
Q

Recombination

A

increases variability

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13
Q

Species

A

a group of individuals that actually or potentially interbreed in nature

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14
Q

Reproductive isolation

A

species become distinct when they no longer exchange genes

-new species arise

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15
Q

Reproductive isolation can occur because:

A
  • They don’t choose to mate with each other or cannot mate with each other (PREZYGOTIC)
  • Or their progeny are sterile or inviable (POSTZYGOTIC)
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16
Q

Biological species concept:

A

members of a species are capable of inter-mating and producing fertile progeny

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17
Q

Prezygotic mechanism

A
  • mechanism before a zygote has formed

- prevents games from 2 different species from fusing and forming a hybrid zygote

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18
Q

Ecological mechanism

A

Prezygotic:

  • Differences in habitat; individuals do not meet thus do not reproduce with one another
  • different ecological niches
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19
Q

Behavioral Mechanism

A

Prezygotic:

differences in mating behavior prevent mating

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20
Q

Temporal mechanism

A

Prezygotic:

-reproduction takes place at different times of the year

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21
Q

Mechanical Mechanism

A

Prezygotic:

Anatomical differences prevent copulation

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22
Q

Gametic Mechanism

A

Prezygotic:
mating between individuals of different species takes place, but the gamete do not form zygotes
-Gametes incompatible or not attracted to each other

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23
Q

Postzygotic mechanism

A

mechanism after zygotes have formed
-gametes of two species fuse and form a zygote, but there is not gene flow between the two species, either because the resulting hybrids are inviable or sterile or reproduction breaks down subsequent generations

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24
Q

Hybrid Inviability

A

Postzygotic:

  • hybrid zygote does not survive to reproduction
  • incompatibility between genomes of the two species preventing the hybrid zygote from developing
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25
Q

Hybrid Sterility

A

Postzygotic

  • hybrid is sterile
  • hybrid embryos compute development but are sterile, so that genes are not passed between species
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26
Q

Hybrid Breakdown

A

Postzygotic

  • F1 hybrids are viable and fertile, but F2 are inviable or sterile
  • closely related spices are capable of mating and producing viable and fertile F1 progeny. However genes do not flow between the few species because of HYBRID BREAKDOWN, in which further crossing of the hybrids produces inviable or sterile offspring
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27
Q

Allopatric speciation

A

geographic barrier initiates speciation by blocking gene flow

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28
Q

Darwins Finches

A

LOOK IN BOOK

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29
Q

Sympatric speciation

A

arises within a single interbreeding population without geographical barriers to gene flow

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30
Q

Races of apple maggot fly

A

ex of sympatric speciation:

  • where resource use is linked to mating preference
  • flies feed on fruits of a specific host tree and mating occurs near the fruits so that larvae can grow up on the ripening fruit
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31
Q

Another mechanism for sympatric speciaiton?

A

hybridization that leads to allopolyploidy

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32
Q

Anagenesis

A

evolution within a lineage overtime

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33
Q

Cladogenesis

A

splitting of one lineage into two

  • once cladogenesis occurs, the branches evolve separately from each other
  • leads to biological diversity since most species exist at the same time
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34
Q

Diversity

A

that different alleles are present at a locus in a population

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35
Q

Classical hypothesis

A

How much heterozygosity is good?

Organisms need low levels of heterozygosity so that they will be well adapted to their environment. Selection favors genotypes that are well-adapted to a specific environment so each organism in a specific environment should had the favorable genotype and there should be little variation in the population.

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36
Q

Balance Hypothesis

A

How much heterozygosity is good?

organisms need high levels of heterozygosity so that they will have the necessary variability to respond to changes in the environment. A successful population would have lots of variability so that it can produce a variety of phenotypes and can allow the population to adapt to a changing environment. Therefore there should be a lot of variability in the population.

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37
Q

Neutralist Theory

A

Why does variation exist?

Many mutations are neutral. This causes polymorphisms to occur in population. Polymorphisms are maintained in the population since neither form has an advantage and the mutant types are not affected by selection.
-eg. two proteins with slightly different amino acid sequences both have proper level of function

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38
Q

Selectionist Theory

A

Why does variation exist?

Many polymorphisms are maintained in the population due to selection. Believe that this will be observed more as we gain more information about the effects of amino acid substitutions.
-Two forms of a protein may allow for optimum performance over a range of cellular conditions.

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39
Q

Which theory is true more of the time, neutralist theory or selectionist theory?

A

Nearly Neutral Model

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40
Q

Phylogenetics

A

study of the relationship among species, individuals, or genes/alleles based on their characteristics

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41
Q

Ways to evaluate evolutionary divergence

A

Morphology
Chromosome Structure
Protein sequences
DNA sequences

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42
Q

Dobzhansky

A

Inversion patterns in D. pseudoobscura correlated with elevation

Different races in Sierra Nevada Mountains:

  • morphology similar
  • Gene order on chromo #3 differs with altitude
  • Gene order differences can be explained by inversion
  • REMEMBER INVERSION HETEROZYGOTE CROSSOVER PRODUCTS ARE OFTEN NOT VIABLE SO SELECTION WILL TEND TO SELECT FOR HOMOZYGOTES
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43
Q

Minimal Mutation Distance

A

minimum total of all necessary nucleotide changes for all amino acids in protein

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44
Q

How do you evaluate changes in DNA sequence

A

RFLPs
Microsatellites
DNA sequence

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45
Q

DNA sequence analysis

A

DIFFERENT PARTS OF THE GENOME EVOLVE AT DIFFERENT RATES

  • 5’ flanking promotor so some sequences will be important for transcription
  • leader and trailer sequences are transcribed, but not translated but may contain signals for RNA processing and ribosome attachment
  • introns removed
  • pseudogenes- don’t code for protein
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46
Q

Nonsynonymous nucleotide substitution vs synonymous

dealing with amino acids

A

NON alter the amino acid

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47
Q

Rates of substitution

A

are lower in amino coding regions of eons but are much higher in nonfunctional DNA such as pseudogenes

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48
Q

Molecular Clock

A

Based on the assumption of constant mutation rate in the change of DNA sequence or amino acid sequence, the difference in sequence between present day organisms can be used to date past evolutionary events

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49
Q

Human Globin Genes

A
  • Multi-gene family

- Evolved by successive gene duplications

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50
Q

OTU

A

Operational Taxonomic Units

OTU can be a species or a strain of a virus or even different alleles within a species

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51
Q

Phylogenetic Trees

A

are used to show degrees of similarity/relationships between OTUs

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52
Q

Unrooted vs Rooted Trees

A

rooted trees have a common ancestor

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53
Q

Terminal Nodes

A

?

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54
Q

Branches

A

?

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55
Q

Internal Nodes

A

?

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56
Q

Constructing Phylogenetic Tress using the parsimony approach

A

-infers phylogenetic relationships based on the minimum number of evolutionary changes in the sequence that must have taken place since the organisms had a common ancestor

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57
Q

Constructing phylogenetic trees using the distance approach

A

-computing differences to infer relationships on overall similarity of organisms, typically by using multiple phenotypic characteristics or gene sequences

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58
Q

Constructing phylogenetic trees using the maximum likelihood/ Bayesian approach

A

infers relationships based on which gives the maximum probability of obtaining the set of characteristics in the organisms

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59
Q

DNA sequence Alignment

A

Identification of HOMOLOGOUS genes and properly aligning their sequences is critical in determine an accurate tree
-typically performed by computers to minimize the number of evolutionary steps

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60
Q

UPGMA

A

Unweighted Pair Group Method with Arithmetic Mean

-relatively simple method of constructing a phylogenetic tree based on computing differences in DNA sequences

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61
Q

Cancer cells

A

have uncontrolled cell division-alteration of cell cycle

Ability to metastasize-spread to other locations

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62
Q

Checkpoints

A

G1 checkpoint
G2 checkpoint
M checkpoint
cdc mutations

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63
Q

G1 checkpoint

A

monitors for proper cell size and undamaged DNA

25% of cell cycle

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64
Q

G2 checkpoint

A

holds up cycle until replication and DNA repair are complete

25% of cell cycle

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65
Q

M checkpoint

A

proper spindle formation and atachment

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66
Q

Protein Kinases and Cyclins

A 
Protein kinases (phospohrlate proteins)
Protein cyclins (Structural protein)

interact to guide progression through cell cycle

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67
Q

Cancer

A
  • mass of tissue cells with unlimited potential to divide/grow and serving no useful function in body
  • Error occurs in cell cycle in 1 cell and increases number of affected cells through mitosis
  • more than 750,000 new cases per year
  • second leading cause of death

CANCER IS GENETIC, BUT CANCER IS RARELY HERITABLE

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68
Q

Hyperplasia

A

uncontrolled cell division

immortal and invasive (immortal in culture, but also invade deeper into surrounding tissues)

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69
Q

Anaplasia

A

Structure/function of cell is undifferentiated

-Tumore has lost the differentiated state and are less similar in structure function and cell type

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70
Q

Metastasis

A

ability to move to and establish tumors at other sites in body

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71
Q

Tumor

A

distinct mass of abnormal cells that do not have normal controls on cell division. NOT EVERY TUMOR IS CANCEROUS

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72
Q

Benign

A

abnormal cells remain localized and do not invade surround tissue

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73
Q

Malignant

A

cancer cells invade surrounding tissue

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74
Q

Metastatic

A

cancer cells spread and establish secondary tumors in other sites in the body

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75
Q

Genetic causes of cancer

A
  • single gene
  • polygenic (more than one gene)
  • chromosome aberratoin (Inversion, TRANSLOCATIONS)
  • Mutations in somatic cell or in gamete producing cell. (if its in gamete producing cell then it ca be passed to next generation.
  • Viruses
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76
Q

Enviromental agents of cancer

A

Carcinogens:

  • can cause mutations
  • can alter gene expression

EX: nicotine, radiation, certain types of plastics

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77
Q

Multi hit model of cancer

A

Most cancers are sporatic and influenced by environment (usually)

  • siblings are rarely affected by the same cancer
  • Populations that migrate to new regions tend to get cancer rates typical of that region indicating factors in the environment are very important, especially diet.

Cancers develop over time (usually)

  • changes in cancer rates due to new environment (ex. smoking) tend to take decades
  • incidense of cancer rises with age

This is consistent with a multi-hit model where cancer arises over time with multiple genetic changes

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78
Q

Pancreatic cancer

A

most deaths per new cases per year. More significant mortality rate.

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79
Q

What are the two types of normal genes that can “go bad” and cause tumor or cancer development

A

Tumor supressor genes prevent bad cells from dividing. Work cell cycle checkpoints and stall the cell to make sure everything is ok, if ok then allow the cell to continue in the cell cycle

Proto-oncogenes allow good cells to divide.

  • Oncogene-when it isn’t work
  • Protogene- good form
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80
Q

Tumor Suppressor Genes

A

Recessive action, have to nock out both to lose function of this gene.

  • normal gene prevents uncontrolled growth
  • abnormal gene- no inhibition- results in tumor if no normal allele present
  • must disrupt both copies of the gene to lose cell cyle regulation (recessive action)
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81
Q

Retinoblastma- RB Gene

A

Inherited Retinoblastoma:

  • An individual inherits an inactivating mutation in one of its RB genes.
  • The other RB gene is inactivated by a somatic mutation during eye development

Sporadic Retinoblastoma

  • An individual inherits two active RB genes
  • Both of the RB genes are inactivated by Somatic mutations during eye development

—40% of cases are inherited (1 bad gene present in zygote)

–Normal protein responsible for regulation at G1 checkpoint

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82
Q

Knudson’s Two hit hypothesis

A

both copies have to be defective in same cell to allow tumor to develop

RB gene

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83
Q

BRCA1 and BRCA2

A

These 2 genes thought to account for about 10% of breast cancers

  • BRCA1-associated with half of hereditary breast cancers
  • 90% of women with mutation in BRCA1 get breast cancer

Strong family history of ovarian cancer may indicate a mutation in one of these genes

Men with these mutations:

  • Have an increased risk of breast cancer (esp. BRCA2)
  • Have increase risk of prostate cancer

Testing by Myriad Genetics:

  • Cost 300-3000$
  • Often covered by insurance

Carriers of a BRCA1 or BRCA2 mutation are much more likely to get breast cancer and ovarian cancer than those who do not have one defective allele.

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84
Q

p53

A
  • chromosome 17
  • Tumor suppressor gene
  • Functions at G1 checkpoint
  • Mutated form seen in diverse cancer types: colon, lung, breast, brain, and is found in altered form in 50% of human tumors
  • The fork in the road: if DNA is damaged, p53 delays cell division until damage is repaired or programs cell to die
  • If p53 not working properly, cell division occur even though DNA is damaged and occurs in unregulated manner
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85
Q

p53 mechanism

A

?

86
Q

Haploinsufficiency

A

When a diploid organism has the loss of function of one copy of a gene and is left with only one functional copy of a gene and this functional copy does not produce enough of the gene product to exhibit the wild-type phenotype.

Although, typically, both copies of a tumor suppressor gene have to be altered to result in cancer, there are some cases where a higher susceptibility to cancer occurs when only one copy of the normal allele is present.

Example: Bloom Syndrome

87
Q

Bloom Syndrome

A

Due to a defective DNA helicase enzyme that is important in repairing double stranded breaks of DNA

Individuals homozygous for mutated BLM gene have a very high rate of cancer

Heterozygous individuals have an elevated risk for colorectal cancer

Similar pattern in mice: Those heterozygous for the BLM were more than twice as likely to develop intestinal tumors than those with two normal alleles

88
Q

Oncogenes

A

PROTO-ONCOGENES: normally promote cell division, but must be activated to regulated properly

Mutation in proto-oncogene results in ONCOGENE that allows uncontrolled cell division

Only need mutation in 1 copy of a proto-oncogene to get tumor whereas in tumor-suppressor gene, both copies must be mutated to get tumor

89
Q

Ras oncogene

A

Cellular Location of Product: Cell membrane

Function of Proto-oncogene:GTP binding and GTPase

90
Q

MYC oncogene

A

Cellular location of product: Nucleus

Function of Proto-oncogene: Transcripton Factor

91
Q

Burkitts Lymphoma

A

Abnormal function of B cells

tumor in the lymph nodes that affect the B cells, therefore can’t produce antibodies

reciprocal transloction between chromosome 8 and 14 places c-myc (oncogene) next to enhancer. Thought that enhancer lead to abnormally high function of c-myc gene.

92
Q

Chronic Myelogenous Leukemia

A

Fatal uncontrolled replication of myeloid stem cells

  • 90% of patients have the philadelphia chromosome
  • Reciprocal Translocation involving chromosomes 9 and 22 places 2 oncogenes near each other.
93
Q

Bcr/c-abl fusion protein

A

Transcription and translation occurs forming the fusion protein. Thought that the protein affects cell cycle control and leads to cancer associated with white blood cells

94
Q

Oncogenes vs Tumor-suppressor genes

A

Oncogenes: Dominant-acting mutation

Tumor-suppressor genes: Recessive-acting

95
Q

Palladin Gene

A

Pancreatic cancer is the 4th leading cause of cancer death even though only about 37,000 new cases occur each year.
- The paladin gene codes for a CYTOSKELETON PROTEIN that is important in maintaining cell shape. Cells that metastasize generally have poor cytoskeleton structure causing them to detach from the tumor mass easily

96
Q

Most cancers are not due to one gene, BUT TO ACCUMUALATIONOS OF MUTations in several genes

A

!!

97
Q

Colon cancer

A
  • Tumor suppressor and oncogenes defective
  • Progression: Benign adenomas to malignant tumors to metastasis

Results in very small polyps and larger polyps

98
Q

Retroviruses

A

can cause cancer by :

  • Mutating and Rearranging proto-oncogenes
  • Inserting Strong promoter near proto-oncogenes
99
Q

Clonal Evolution

A

Over time, tumor cells acquire more mutations that allow them to be progressively more aggressive in proliferation.

These mutations can affect:

  • Cell cycle regulation
  • Signal Transduction
  • DNA Repair
  • Telomere Length
  • Chromosome segregation (many tumor cells are aneuploid)
  • Vascularization
100
Q

Signal Transduction:

A

external signal triggers a cascade of intracellular reactions to produce a specific response

101
Q

Ras Signal-Transuction pathways

A

Stimulates the cell cyle

102
Q

Defective Nucleotide Excision Repair

A

Xeroderma Pigmentosum

103
Q

Defective Mismatch Repair

A

Colorectal, endometrial, stomach cancers

104
Q

Role of Telomere Length in Cancer

A
  • Typically Telomeres shorten as a cell ages and this ultimately contributes to the death of the cell
  • Normally, telomerase works in germ line cells, but not in somatic cells: allows somatic cells to die, be replaced, etc.
  • Tumor cells often have telomerase expression, which is thought to contribute to the “immortality” of cancer cells.
  • Mutations in genes that regulate telomerase activity may be important in cancer, but role of telomerase is not clear at this time.
  • Possible cancer therapy could be to block telomerase activity
105
Q

Cancer cell kayotypes

A

abnormal

106
Q

Angiogenesis

A

(growth of new blood vessels) is important to tumor progression

  • Growth factors and other proteins involved in angiogenesis are often over expressed in tumor cells
  • Angiogenesis inhibitors may be inactivated or under expressed
107
Q

Is it possible to fight to cancer by preventing angiogenesis?

A

Metastasis is the cause of death in90% of human cancer cancers

108
Q

Classic Cancer Model

A
  • All tumor cells can from new tumors and are therefore equally tumorigenic
  • Unregulated growth is due to a serial acquisition of genetic events leading to the expression of genes that promote cell proliferation while silencing the growth of inhibitory genes and blunting cell death
  • Cancer is a proliferative disease
109
Q

Cancer Stem Cell Theory

A
  • Tumors arise from “cancer stem cells” (CSCs) that are SELF RENEWING and MULTIPOTENT
  • CSCs persist in tumors and may be the cause of relapses and metastasis
  • This might explain why many cancers are difficult to treat
110
Q

Chemotherapy

A

may kill the bulk of tumor cells, but may not affect the CSCs, leading to a recurrence of the cancer
-Development of specific therapies targeting CSCs may provide hope for increased survival and quality of life especially for metastatic disease.

111
Q

Restriction Enzyme

A

?

112
Q

Palindrome

A

?

113
Q

Joining TWO DNA molecules

A

1) digestion with HindIII (or other restriction enzyme) causing 5’ and 3’ sticky ends
2) Nick in sugar phosphate backbone joining the two different strands
4) Ligase to repair gaps

114
Q

Cloning Vector

A
  • Vector is a carrier DNA
  • Molecule that is capable of independent replication into which a DNA fragment can be cloned. Its purpose is to carry foreign DNA into the cell
115
Q

What does the ideal cloning vector contain?

A

1) origin of replication
2) Selectable/screenable markers
3) A single cleavage site for each restriction enzyme

116
Q

Plasmid Vectors

A

can carry 100 bp- 10kb insert (usually 6-7 kb)

117
Q

What does plasmid vectors contain?

A

need origin of replication

need 2 marker genes

118
Q

Lambda virus as a vector

A
  • Lambda can be lytic or lysogenic: We want LYTIC since want to replicate inserted sequence
  • DNA is made as a CONCATAMER (many copies joined together)
  • It is cut to proper size at locations called “cos” sites. Cos sites are important in proper packaging of DNA into viral head
  • DNA is packaged as a piece 45 kb long and size is critical in packaging.
  • To use lambda as a vector, remove lysogenic genes (15kb) in center and ad 15 kb insert- size is critical!
  • Virus containing recombinant molecule injects DNA into cell and can replicate
119
Q

Cosmids

A
  • Contains cos genes from lambda ask that it can be packaged into a viral coat and get into the bacterial cell like a virus
  • Contains origin of replication for bacteria so that it replicates like a plasmid in bacterial cell
  • polycloning site
  • selectable marker (amp^r)
  • Holds about 44kb insert (big piece)
120
Q

Expression vectors

A
  • allow the inserted gene product to be produced
  • Must contain sequences required for transcription and translation of the gene in addition to other vector characteristics
121
Q

Expression vectors contain:

A

-operon sequeces that allow inserted DNA to be transcribed and translated.
-Include sequences that regulate- turn on or turn off- the desired gene
-

122
Q

Bacterial Artificial chromosome

A
  • holds about 300 kb insert

- uses F factor genes for replication

123
Q

Yeast Artificial Chromosome

A
  • Yeast telomere and add centromere regions
  • selective markers
  • origin of replication
  • restriction sites for cloning
  • can hold BIG pieces of DNA (several hundred kb)
124
Q

Shuttle Vectors

A
  • can replicate in 2 or more host organisms
  • eg. Yep24- replicates in yeast and e. coli
  • contains selective markers for E. coli and yeast and sequences which allow replication in both hosts
125
Q

Insertional Inactivation

A

The inserted DNA inactivates a gene in the vector by inserting into that gene. This allows cells which contain the recombinant DNA molecule to be identified.

126
Q

Identification of different cell types

A

cells with no uptake, cells that took up the original vector and cells that took up the recombinant plasmid. In this example, the cells that took up the recombinant plasmid will be ampr, tets. There was insertional inactivation of the tetracycline resistance gene

127
Q

Ligation experiment

A

conducted to join foreign DNA to vector. The foreign DNA and the vector are both cut with the same restriction enzyme. The DNA’s are mixed and DNA ligase is added. Some recombinant molecules should form.

128
Q

Polylinker

A

DNA sequence within marker gene that contains unique recognition sites for many restriction enzymes into which foreign DNA can be inserted

129
Q

Transformation experiment

A

Conducted to allow cells to take up products from ligation experiment

Products from ligation are put into bacteria
Then cells containing recombinant plasmid are identified: use of marker genes

130
Q

Selectable/Screenable Marker

A

gene (on vector) that can be used to differentiate between cells with and without the vector or that can be inactivated by insertion to differentiate between cells that do or do not have the insert

131
Q

Cells that take up nothing or take up only foreign DNA are?

A

amp^s and tet^s

132
Q

Cells that take up original vector are?

A

amp^r and tet^r

133
Q

Cell that take up recombinant plasmid are?

A

amp^r and tet^s

134
Q

Blue-white Screening

A

?

135
Q

Library

A

-set of clones derived from 1 approach

136
Q

Type of libraries

A

Genomic

Chromosome Specific:: use micro dissection or flow cytometry to isolate a specific chromosome

cDNA libraries:

  • cDNA=DNA that is a copy of mRNA
  • only biologically active genes
  • cDNA is already properly processed for expression
137
Q

Reverse Transcriptase

A

RNA dependent DNA polymerase

-used to make cDNA

138
Q

isolating mRNA

A

1) a specific column contains short olgio (dt) chains linked to cellulose
2) Total cellular RNA is isolated from cells and passed through the column
3) The poly(A) tails of mRNA pair with oligo(dT) chains and the mRNA is retained in the column where as the rest of the RNA passes through
4) The mRNA is washed front he column by adding a buffer that breaks down the hydrogen bonds between the poly(A) tails and the oligo(T) chains leaving only mRNA with poly (A) tails

139
Q

making cDNA after isolating mRNA with poly(A) tails

A

1) oligo (dT) primers anneal to the poly(A) tails of the mRNA and provide 3’ -OH groups for DNA synthesis
2) Reverse Transcriptase synthesizes a DNA strand by using the mRNA as a template.
3) The RNA-DNA hybrid molecule is briefly treated with RNase, which partly digest the RNA strand
4) DNA polymerase synthesizes the second DNA strand by using the short undigested RNA pieces as primers and the nicks in the sugar-phosphate backbone are sealed by DNA ligase

140
Q

Screening a library

A

1) a disc of nitrocellulose or other membrane is laid on top of the bacterial colonies
2) A few cells from each colony adhere to the nitrocellulose filter
3) The cells are disrupted, and their DNA is denatured and fixed to the filter
4) A labeled probe hybridizes with any complementary DNA
5) Excess probe is washed off and the membrane is overlaid with X-ray film which detects the presence of the probe
6) Comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest

141
Q

In situ Hybridization to locate Specific sequence

A

?

142
Q

PCR

A

Polymerase Chain Reactoin

  • Denature DNA by heating to 95 Celsius (breaks hydrogen bonds and allow strands to separate)
  • Each strand serves as a template for replication
  • Primers anneal (50-65 C) to identify target that will be amplified
  • Taq polymerase adds nucleotides to 3’ end of primer (72 C). Repeat many times
143
Q

Kary Mullis

A

Developed PCR protocol

144
Q

Thermal Cycler

A

machine that is programmed to alter temperatures so that PCR can be conducted

145
Q

Limitations of PCR

A
  • Must know something about sequence surround gene of interest in order to use PCR to clone a gene
  • PCR reactions are easily contaminated from other DNA in the lab
  • Taq polymerase does not proofread and correct errors (error rate about 1 in 20000BP)
  • Fragments amplified by PCR are relatively small
146
Q

Electrophoresis

A

separating out pieces of DNA based on size

  • Distance migrates is inversely proportional to the log of the fragment size
  • DNA loaded at negative pole and migrates to positive pole “RUN to RED”
  • Small fragments migrate faster than large fragments
  • Sizes can be determined based on comparing fragment migration to that of a control of known size
147
Q

Blotting

A

process of transferring molecules that were previously separated (on a gel, etc.) to a membrane that is better able to support additional testing

148
Q

Southern Blot

A

DNA fragments separated based on length

149
Q

Northern Blot

A

RNA fragments separated based on length

150
Q

Western Blot

A

Proteins that are separated on molecular weight, isoelectric point, electric charge, etc.

151
Q

Southern Blot Process

A

1) Single Stranded DNA fragments from a gel are transferred to nylon membrane using capillary action in the Southern Blot
2) The nylon membrane is incubated with labeled, single stranded probe DNA
3) The probe binds to complimentary DNA fragments on the nylon
4) The position of the probe is identified

152
Q

Probe

A

Single Stranded DNA that is a sequence we are interested in

153
Q

Dideoxy Sequencing Reaction

A

DNA Sequencing!
Developed by Sanger
- DNA replication reaction proceeds until a dideoxy nucleotide is incorporated
-No further extension occurs
-Detect at which position the dideoxy nucleotide was incorporated.

154
Q

Reporter

A

Foreign Gene is used as a reporter- on or off but can detect when it is on

155
Q

GFP

A

Reporter GENE

  • GFP linked to a protein allows us to visualize when protein is expressed in living cells over time
  • GFP=Green Fluorescent Protein (from a jellyfish)
  • GFP gene is joined to protein gene
  • Gene is put into organism
  • Organism produces a fusion protein
  • Shine UV light on organism
  • GFP fluoresces
  • Allows expression of protein to be seen over time in living organism
156
Q

Therapeutic proteins

A
  • Production of Eukaryotic Proteins

- Human growth hormone, insulin, blood clotting factors

157
Q

Industrial Applications

A
  • Production of Eukaryotic Proteins
  • Rennin In cheese manufacturing
  • Enzymes such as bacterial proteases in detergents and meat tenderizers, amylases to degrade complex sugars
158
Q

Transgenic Plants and animals

A

Transgenic plant or animal is one in which a foreign or exogenous gene has been introduced into its genome, thereby altering its genetic constitution

159
Q

Ti Plasmid

A

Ti plasmid is a tumor inducing plasmid from Agrobacterium tumefaciens. It causes crown gall disease in the normal infection process
-The Ti plasmid inserts into the plant chromosome to cause the disease

Goals:

  • Remove tumor causing genes
  • Leave transfer functions intact
  • Harness the transfer functions so that the plant will be “infected” with a gene of interest
  • Allow inserted gene to function in plant
160
Q

Transposon

A

mobile piece of DNA flanked by terminal repeat sequences and typically containing genes coding for transposition (jumping genes)

161
Q

Luciferase gene from firefly

A

emits a glowing color

  • attempted to put into tobacco Plant
  • T-DNA + insert inserted into 1 plant chromosome-heterozygous plant that glows

**Mendelian Inheritance pattern of 3 glowing: 1 non-glowing

162
Q

Why do we use luciferase gene from the firefly?

A

Its used as a reporter gene so that if place regulatory sequences upstream form laciferase gene, regulation of luciferase can be monitored during developement

163
Q

Forward genetics

A

start with a mutant phenotype and seek out the gene that causes that phenotype
-characterize that mutant type- is it dominant, recessive, etc.

164
Q

Reverse Genetics

A

Start with a DNA sequence ( a genotype), alter its function or prevents its expression and observe the effects on the phenotype.

165
Q

Transgenic mice

A

useful for forward vs reverse genetics

  • Gene Function Studies
  • Inject gene of interest into fertilized egg
  • Implant in female
  • Test progeny for presence of gene
  • Mate to obtain mice homozygous for gene
  • Study gene function
166
Q

Knock-out mice to study Gene function

A
  • insertion of neo gene into another gene will inactivate the other gene and provide resistance to the antibiotic G418
  • injected altered gene into embryonic stem cells and select stem cells that have taken up gene
  • Inject those cells into early mouse embryos. Isolate mice that show sectors with injected cells (black coast stem cells are often used as a visible marker
  • Mate to get homozygous knock-out mice. Phenotype of mouse identifies gene function
167
Q

RFLP

A

Restriction Fragment Length Polymorphism

  • coexistence of two or more restriction fragment mapping patterns revealed by hybridization to a single probe
  • Location of probe relative to the variable cut site is important in determining fragment sizes that will be seen.

-RFLP pattern within gene correlates with genotype in pedigree

168
Q

Gene Therapy

A
  • Introducing function copies of a gene into individuals who have only defective copies of that gene.
  • In successful gene therapy, the transgene will make the missing gene product and restore normal phenotype
  • Somatic Cell (non-heritable): treats, but does not cure the disease. All current gene therapies are somatic cell therapies
  • Germ-line (heritable): major moral and ethical considerations
169
Q

When introducing normal genes into cells this requires?

A

the use of viruses:

  • Retroviral vectors integrate into the DNA of the host cell
  • Transgene is transmitted to all progeny cells in the cell lineage
  • Transgene may integrate so that it disrupts function of another gene
170
Q

Transgene

A

introduced copy of the gene

171
Q

NIH requirements for Gene Therapy

A
  • Gene must be cloned, well characterized and available in pure form
  • An effective method must be available for delivering gene to desired cells and/or tissues
  • The risks of gene therapy to the patient must have been carefully evaluated and shown to be minimal
  • The disease cannot be treatable by other methods
  • Data must be available from preliminary experiments with animal models or human cells and must indicate that the proposed therapy should be effective
172
Q

PCR as diagnostic Tool’s Goal

A

determine if a particular DNA sequence is present in a sample of DNA
EX: is the blood sample infected by HIV

173
Q

PCR as diagnostic tools method

A
  • conduct PCR of a blood sample using primers for known HIV sequences
  • Separate fragments on gel and determine if they are the proper length of the known HIV fragments
174
Q

Real-Time PCR

A
  • Quantifies the amount of nucleic acid present after each cycle of PCR
  • Uses a fluorescent probe to identify the DNA of interest
  • *Can be combined with reverse transcription to quantify mRNA (or other RNAs) and evaluate gene expression and the amount of mRNA produced under variable conditions

EX: does a drug affect production of a particular mRNA?
-Compare the amount of mRNA produced by cells that are treated with a drug and control cells

175
Q

STR and VNTR

A

Short tandem Repeats
Variable Number of Tandem Repeats Micro satellite regions
-The allele is based on the length of the DNA segment with different alleles having different lengths because they have different number of copies of a short repeated DNA sequence
-Useful loci for paternity/forensics purposes must be polymorphic (many forms)

176
Q

CODIS

A
  • Combined DNA index System
  • DNA databases funded by FBI
  • Uses 13 polymorphic regions used for forensic identification

-3 tiered system: separate federal, state, and local databases

177
Q

CODIS:

Where do the DNA profiles come from?

A
  • more than 9.4 million come from offenders
  • about 360,000 from crime scenes
  • DNA from missing person, relative of missing persons and unidentified human remains
178
Q

STR analysis

A

Power of STR analysis:
-Ability to use small amounts of forensic material
-Statistical power of discrimination
13 core STR loci that are currently used for discrimination

These assort independently so product rule applies regarding probability of a particular combination of alleles

  • However, accuracy of probabilities does depend on accurate probability estimates for particular alleles in the population
  • Typically estimates are about 1 in 1 billion
  • Data for one population is not necessarily correct for another population
179
Q

Capillary Electrophoresis

A
  • Same principle of separating DNA fragments based on their size
  • Easy to test multiple genes in one automated run
180
Q

FBI database

A
  • if you were detained for some federal offenses, your DNA sample can be taken and put in the DNA database- if four not guilty can petition to get it removed
  • If you are detained as immigrant (illegal or not), your DNA sample can be taken and put in the DNA database
  • They do no need a warrant (in some states) to collect DNA from suspects
181
Q

Ethical Issues with GN testing

A

Use of PARTIAL MATCH DATA: search databases for partial matches to help identify suspects

  • Familial DNA testing
  • Conflict betweens solving crimes and protecting privacy

There have been 311 people who were falsely convicted released from prison

182
Q

Genetics

A

study of one or a few genes at a time

183
Q

Genomics

A

Study of whole genomes- all of the genes, the interaction between genes and interactions with the enviroment

184
Q

Genome Science

A

Goal is to study the structure, function and evolution of whole genomes. This complements the study of quantitative molecular and developmental genetics

185
Q

Genome-Wide Association Studies

A

GWAS

  • studies that look for non-random association between the presence of a trait (phenotype) and alleles at many different loci scattered across the genome
  • Especially useful for identifying QTLs-those loci that are important in quantitative traits
  • Find association between molecular marker and look for candidate gene can affect a that that located near the marker
186
Q

DGRP

A

Drosophila Genetic Reference Panel

  • IS AT NCSU
  • 205 fully sequenced inbred lines from a natural population
  • Used to identify QTLs
187
Q

Mouse Collaborative Cross

A

Started with 8 inbred mouse lines:
-3 wild-derived + 5 lab lines

Mated to randomize genes resulting in about 1000 inbred lines that are mixtures of the 8 original lines

188
Q

What could genomics mean for you?

A

Personalized medicine that takes into account the molecular events underlying a disease and your “genotype” to determine the best treatment option for you

  • we know that not everyone responds the same to a particular medicine or therapy
  • Tailoring medicine to individuals should provide better medical care and prevent harmful side effects

EX: tamoflaxin, some anesthetics

189
Q

Warfarin

A

Blood thinner

  • widely prescribed anti-coagulant (coumadin- prescribed in the from called)
  • Greater than 10 fold inter-individual variability in the dose required to attain a therapeutic response
  • Required dose also varies depending on individuals diet and is not the same for each person

2 genes influence effect dose

  • CYP2C9 (warfarin metabolic enzyme)
  • VKORC1 ( vitamin K epoxide reductase complex 1)
190
Q

SNP

A

Single Nucleotide Polymorphism

  • A SNP is a specific site in the genome where the DNA base varies in at least 1% of the population
  • There are about 10 million SNPs in the human genome
  • SNPs located near each other in the genome are often inherited together and can often be grouped as a HAPLOTYPE (a sequence of SNP patterns along the length of a chromosome)
191
Q

Linkage Disequilibrium

A

The non-random association of certain variants with each other so that these combinations occur more frequently in the population than expected based on independent assortment

192
Q

TagSNPs

A

Linkage Disequilibrium
tagSNPs= the few SNPs used to identify a haplotype
-A haplotype of 1000s of SNPs can be identified by only a few SNPs
-Thought that about 100,000 SNPs can be used to identify most haplotypes in humans

193
Q

Using SNPs in association studies

A

Correlate Presence/absence of specific SNP or SNP haplotype with presence/absence of genetic disorder

Advantage:
-SNP association studies identify DNA difference between individuals more quickly/more easily that sequencing entire genomes

Problem:

  • Correlation is not equal to CAUSATION ( but is a starting point for research)
  • It takes a lot of research to identify if a particular SNP is meaningful for a specific disorder
194
Q

Ethical Questions regarding genomics

A
  • How do you control/regulate the spread of use of your genetic information
  • Is the information going to make life simpler or more complex?
  • Who has access to your genetic information?
195
Q

GINA

A

Genetic Information Nondiscrimination Act

  • Health insurance companies cannot decrease coverage or increase rates based on rates of genetic tests
  • Employers cannot discriminate (hire/fire, etc.) based on results of genetic tests
  • Neither Health insurance Companies or employers can require genetic testing
196
Q

Mapping, Sequencing and analyzing the structure and function of whole genomes involve:

A
  • collecting sequence data
  • Correlating genetic, cytological and physical chromosome maps
  • identifying DNA sequences that contain genes of interest in the genome and analyzing the function of those gene products
197
Q

What companies are doing Direct to consumer testing?

A

23 and me
deCODE me
Navigenics

198
Q

Genetic map of a chromosome

A

constructed from recombination frequencies in units of CENTIMORGANS. Includes RFLP sites

199
Q

Cytological maps of a chromosome

A

Banding patterns based on chromosome staining

200
Q

Physical maps of a chromosome

A

molecular distance in bp,kb, or mb
-physical maps often show restriction enzyme recognition sites, locations of particular clones or sequence-tagged sites (STS)

201
Q

Anchor Markers

A

used to correlate different maps

-are mapped both genetically and physically so can be used to correlate the genetic and physical maps

202
Q

STS

A

Sequence tagged sites
-short, unique DNA sequences (200-500 bp) used to link physical and genetic maps. These sequences are hybridized to overlapping clones on physical maps and are hybrized to cytological maps using in situ hybridization to anchor map types

203
Q

EST

A

expressed-sequence tags
-short cDNA sequences that are used to link genetic and physical maps. These cDNA sequences are used as hybridization probes to anchor maps

204
Q

Contig

A

overlapping sets of clones that give a physical map of part of a chromosome

205
Q

Positional Cloning

A

isolating a clone of a gene by searching its general position in the genome

206
Q

Human Genome Project

A

1988-congress funds NIH and DOE
1991- project begins
2001- first draft published (90% complete)
2003- second draft compete (99% of euchromatin)

Project still continues with focus on variation

Craig Venter and FRANCIS COLLINS

207
Q

What are the two approached to the human genome project

A

1) Map based sequencing:
- Make genomic library
- Fingerprint library and orient clones relative to each other
- Screen the library with markers that allow you to relate it to the recombinant map
- Map based sequencing relies on detailed genetic and physical maps to align sequence fragments

2) Whole genome Shotgun approach
- Small-insert clones (plasmids) prepared directly from genomic data and sequenced
- computer programs assembly by examining overlap
- Advantage of being highly automated

208
Q

Orthologs

A

homologous sequences found in different species

209
Q

Paralogs

A

homologous genes int eh same species and arrive through gene duplication

210
Q

Microarray Analysis for Gene expression

A

allows study of gene expression including ability to quantify expression level between issues or strains of organism

211
Q

Microarray analysis of RNA from cancer and Noncancerous cells

A

Red-higher expression in cancer cells
Green: higher expression in non cancer cells
Yellow: equal gene expression in both cell types

212
Q

Bioinformtics

A

combines computer science and biology

Goals include:

  • Maintaining and analyzing data bases of sequence data
  • Comparing structural and functional features of DNA sequences and resulting proteins EX: BLAST
  • determining evolutionary relationships among genomes

GN 311 (17 decks)

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