final biotech sharp

exam 4

exam 4

forensic science

law and science

historical examples of forensic science

photography (1800s) and forensic science (1900s)

who invented dna fingerprint

sir alec jeffreys in 1985

benefits to using dna for crime scenes

- everyone has unique alleles

- structure of DNA is same but order is diff

- identifiable organism bc you have a complete set of DNA

what % of dna differs from person to person

1/10 of 1%

- or 3.2 mil nucleotide differences

what did alec jeffreys discover

- invented RFLP

- discovered VNTRs

- discovered VNTR's while using RFLP to analyze DNA

- unique signature found in each person's genome

- depends on a small portion of genome

what is rflp?

Restriction Fragment Length Polymorphism

- dna fingerprinting technique that involves DIGESTING DNA into FRAGMENTS of diff lengths by using RE's

what are vntr's

variable number of tandem repeats

- NON-CODING regions of DNA that REPEAT the same sequence over and over

how to do rflp

SOUTHERN BLOT

most RFLP markers are _____ ;_____

di-allelic

co-dominant

RFLP inherited from

mom and dad

RFLP is _____ locus specific

HIGHLY

what type of paper used in southern blot?

nitrocellulose filter paper

how can DNA profiling benefit us?

- facilitate police detective work

- identify human remains of disaster victims

- clarify paternity disputes

- clarify immigration disputes

- study of animal and plant population genetics

- revolutionized the fields of zoology, botany, and agriculture

-track and confirm organisms that spread disease

-determine mystery meats

- confirm poaching of endangered species

-medical diagnostics

two types of forensic DNA testing are?

- rflp

- pcr used with vntr

pros and cons of RFLP

CON

- requires larger amounts of DNA

PRO

- DNA cant be degraded

- no prior sequence info is required

pros and cons of PCR used w VNTR

PRO

- requires much less DNA

- DNA can be partially degraded

CON

- extremely sensitive to contaminating DNA

- must have prior sequence info

VNTRs are what?

-repetitive DNA present in all mamalian genomes

-highly polymorphic w large # of alleles

microsatellites/short tandem repeats (STRs)

- repetitive DNA between 2-6 base pairs

- repeated typically between 5-50 x

- inherited from mom n dad

- comprise 3-5% of mammalian genomes

type of slippage during replication: a mechanism for STR mutation

1. no mutation

lines parallel

2. expansion

shaded has no loop

unshaded has loop

3. contraction

shaded has loop

unshaded has no loop

FBI has chosen 13 unique _____ for analysis and comparison in its library of _______.

1. STR's

2. DNA fingerprints

what are the odds that 2 individuals will have the same 13-loci DNA profile?

more than one billion

as of 2007, how many loci does the FBI require?

20 loci

extraction of DNA can be purified _______ or ________

CHEMICALLY (detergents) or MECHANICALLY (pressure to force DNA out of cell)

what are the enemies of evidence?

- sunlight and high temp

- bacteria

- moisture

Best sample from suspect DNA is fresh, _____, ______

blood; leukocytes

PCR and DNA fingerprinting

- amplifies DNA found at crime scene to amount that can be analyzed

- DNA primers for the flanking regions of CODIS sites results in DNA amplification at specific STR sites

- one of 2 primers must be fluorescently labeled

STR analysis is the process in which?

- STR's are amplified by PCR

- alleles separated and detected using CAPILLARY ELECTROPHORESIS

- this allows # of repeats in each of the 2 alleles on a homologous chromosome to be determined

what is done with the evidence collected at crime scene?

compared with evidence collected from a database, tests based on exclusion

first reported use of genetic fingerprinting in a criminal case was when?

1983 for the Naborough Village murders

techniques in World trade center attack?

STR, mtDNA, SNP analysis

South tsunami techniques?

mtDNA, y-STR's

human error and sources of contamination

- custody of samples is compromised

- Collection of evidence must be systematically recorded

- access to evidence must be controlled

- Follow standards of lab practice and procedures to prevent DNA damage during the analysis

DNA and juries

-must make sense to the jury.

-statistics can be confusing

-"1 chance in 50 billion" may lead to focus on the one possibility and discounting the odds against it happening.

paternity testing

analyze samples from child and adults involved

in order to check for paternity, its possible to draw fetal cells from what?

amniotic fluid

mitochondrial DNA is used to

-examine samples that CANT be analyzed thru RFLP or STR

- inherited from mother only

- 100-fold higher mutation rate

Nonhuman DNA analysis:

ginseng in america

ginseng in asia

- boost energy

- decrease stress

Nonhuman DNA Analysis: prove a hunter killed a bear illegally

- blood samples from den were taken, and matched

- eye witness

-bears premolars were extracted to confirm sex and age of bear

many important genes are highly conserved from species to species based on DNA sequence called?

homologs

study of genes involved in apoptosis in _______ helps scientists understand human neurodegenerative disease

apoptosis

Genes determining human body plans, organ development, aging and death are virtually identical to those in _______

fluit flies

we can use model organisms to...

- identify disease genes

- test gene therapies

- develop drug-based therapeutic approaches

- determine the effectiveness of gene therapies

- check safety in preclinical studies

the Ob gene is found in mice and humans codes for the protein ________.

leptin

- produced by adipocytes

-regulates HUNGER

fI Ob gene is nonfunctional and leptin is not produced, mice and humans become _________.

OBESE

biomarkers for disease detection is critical for providing the best treatment and improving the odds of survival... what is it?

measurable indicator of some biological STATE or condition;

typically proteins produced by diseased tissue or proteins whose production is increased when a tissue is diseased

(ex: PSA, prostate-specific antigen)

biomarker examples?

-Salivary cortisol-Body mass index

-Activity monitor-Blood pressure

-Glucose testing

-Pulse oxygen

-Heart rate

chorionic villus sampling

fetus is 8-10 wks

- REMOVE small portion of a layer of cells called the CHORIONIC VILLUS that helps form the placenta

- create karyotype

amniocentesis

fetus is 14-16 wks

- take a sample of AMNIOTIC FLUID

- create a karyotype

what 2 exams test for chromosome abnormalities?

chorionic villus sampling and amniocentesis

FISH (Fluorescence in situ Hybridization)

updated technique for karyotyping,

useful for identifying missing chromosomes, extra chromosomes, or defective ones

Most genetic diseases result from mutations in specific genes; can easily detect with:

RFLP or ASO

Allele specific oligonucleotide analysis (ASO)

allows for the detection of single nucleotide changes even if the mutation does not change a restriction site

how to use RFLP to determine sickel cell

is they have DIFFERENT sizes on autoradiogram, its sickle cell disease

if it has BOTH, they are a carrier.

how to use ASO to determine sickel cell disease

A = normal allele

S =. sickle cell allele

homozygous - A:light S:dark

heterozygous - A+S:same color

preimplantation genetic testing

- ASO

- PCR

- FISH

used to screen for defects in single cells from 8 to 32 cell stage embryos created by IVF

SNPs (single-nucleotide polymorphisms)

- most common form of genetic variation

- make up 9% of human genetic variation

- causes change in prot structure that influences traits in many ways/ produces disease

- can predict susceptibilities to: stroke, cancer. etc.

DNA microarray

- glass microscope slides spotted with DNA probes representing genes

- can be used to screen a pt. for a particular set of alleles (that might be expressed in a particular disease condition

RNA microarray

use a patients transcriptome which include all mRNA in population of cells

oncogenes

genes involved in growth of cancer cells

tumor SUPPRESSOR genes

produce prots that keep cancer formation in check

gleevec

first pharmacogenomic in 2001

- targers BCR-ABL fusion protein that causes chronic leukemia

pharmacogenetics

designs the most EFFECTIVE drug therapy and treatment strategies BASED ON specific GENETIC PROFILES of a patient

why do different degrees of effectiveness and side effects occur with pharmacogenetics?

genetic polymorphisms

what do drugs in chemotherapy do?

- target RAPIDLY DIVIDING cells

- affect normal body cells such as hair, skin, bone marrow cells that regularly divide

BRCA1 an BRCA2

breast cancer 1 and 2

tissue from breast tumor can be used to isolate what?

RNA or DNA for SNP and MICROARRAY analysis

- this can determine which genes are involved in a woman's breast cancer

cancer vaccines

injected with cancer cell antigens to stim immune system to attack cancer cells

-not preventative treatment

monoclonal antibodies

purified antibodies that are specific for certain molecules

(ex. lymphoma, cancer, alzheimers, drug addiction)

production of monoclonal antibodies (Mabs) used to treat

cancer, heart disease, transplant rejection

- process to produce desired antibody

- spleen cells

direct capture/direct detection

primary antibody conjugate

direct capture/indirect detection

-primary body

-secondary body conjugate

indirect capture/indirect detection

super specific for antigenic material

"capture assay" sandwich

gene therapy

delivery of therapeutic genes into the human body to correct disease conditions created by a faulty gene (or genes)

how is gene therapy done?

-ex vivo gene therapy

-in vivo gene therapy

ex vivo gene therapy

- cells removed from patient

- treated w techniques similar to transformation

- then reintroduced to the person

in vivo gene therapy

- introducing genes directly into tissues and organs in body

- challenge:delivering genes to only intended tissues and not tissues throughout the body

vectors for gene delivery

typically rely on viruses as vectors

- use viral genome to carry a gene and use virus itself to infect human cells, which includes the gene

EX: flu, herpes virus

other gene delivery options?

naked DNA and liposomes

first human gene therapy

SCID patient in 1990

SCID (severe combined immunodeficiency)

defect in gene called ADENOSINE DEAMINASE (ADA)

regenerative medicine

growing cells and tissues that can be used to replace or repair defective tissues and organs

what are stem cells

-two layers to the blastocyst

-trophoblast is outer layer, forms placenta

-inner cell mass is the source of human embryonic stem cells

hESCs

Human embryonic stem cells

- PLURIPOTENT cells derived from the inner cell mass of a blastocyst.

TOTIPOTENT stem cells

capable of giving rise to any cell type or a complete embryo

PLUROPOTENT stem cells

- capable of developing into any cell type or tissue except those for a placenta or embryo

MULTIPOTENT stem cells

- can give rise to many but limited types of cells

- found in adult tissues

- have ability to self-renew

exam 3

exam 3

what are microbes

tiny organisms

bacteria, fungi, protozoa, algae

what percent of all bacterial species have been identified cultured and studied; and estimated to comprise what percent of the earths biomass?

1%

50%

bioprospecting

discovery and development of new products from biological resources

microbial enzymes function

application

from food prod to research

3 microbial enzymes?

1) taq DNA polymerase

2) Cellulase

3) Subtilisin

yeast two hybrid system

determines if prots A and B interact

in yeast 2 hybrid system, what are the two FUSION PROTEINS created?

- protein a: THE BAIT with DBD (DNA binding domain) of transcription factor that binds to promoter

- protein b: THE FISH with AD (activator domain) that binds to the enhancer

If protein "A and B" interact what happens to the DNA binding domain and the activator domain?

DBD and AD brought together

main purpose of yeast two is to test interaction of diff _____ molecules

protein

what does BAIT plasmid have?

GENE OF INTEREST

what does FISH plasmid have?

a gene that causes a fusion of Protein "B" (the fish) with an activator domain (AD) that binds to the enhancer