What is molecular biology?
The branch of biology that deals with the structure and function of molecules essential to life
What is biotechnology?
The use of biology to create useful technologies and products
Biotechnology in forensics
The use of techniques from molecular biology to analyze biological evidence -blood, saliva, skin cells-extract DNA profiles
Matthias Schleiden and Theodor Schwann
all organisms are made of cells and cells are the basic unit of life
Gregor Mendel deciphered
the genetic laws by conducting experiments on pea plants, and discovered the basic (segregation and assortment)
-dominant and recessive traits
Friedrich Miescher isolates
Nuclein from white blood cells
Walter Sutton and Theodor Boveri proposes that hereditary factors
are located on chromosomes which segregate during cell division
P.Levene, W.Jacobs, and others demonstrate
that RNA is composed of a sugar(ribose) plus four nitrogen bases, and that DNA contains a different sugar (deoxyribose) plus four bases
Frederick Griffith performed
information in the bacterium Streptococcus pneumoniae
-Heat killed bacteria transfer traits to live bacteria
-Evidence of a transforming principle
How did griffith perform his experiment?
Injected living S cells (in capsule) = mouse died
Inject living R cells (not in capsule) = mouse alive
Heat killed S cells (in capsule but non-pathogenic
Mixture of heat killed S cells and living R cells= mouse dies
Avery, MacLeod, MacCarty used a similar transformation test
injected DNase, RNase, lipase, protease, and other and determined that DNA is the transforming principle
Hershey and Chase shows that
DNA is transferred from bacteriophages to bacteria
Francis Crick lays out the
central dogma
DNA-> RNA-> Proteins
Two main types: of nucleic acids
DNA: carries genetic information in living organisms
RNA: plays various roles in coding, decoding, regulation, and expressino of genes
What do nucleotides contain?
- a nitrogenous base (adenine, cytosine, guanine, thymine, uracil)
- phosphate group
-linked to the 1C and 5C position of a (deoxy)ribose sugar
Which nitrogenous bases are purines and pyrimidines?
-Adenine and guanine are purines
-Cytosine, thymine, and uracil resemble pyrimidine
Bond that holds the nitrogenous base
glycosidic bond
The OH on the 3' carbon attacks
the phosphodiester bond and releases 2 phosphate groups
DNA double helix structure
Has sugar-phosphate backbones on the outside and the bases are aligned to the interior
When melting DNA,
The hydrogen bonds that hold the DNA strands together will weaken and break and causes denaturation
When annealing,
DNA is cooled to allow for the strands to come back together
Chromatin organization: the nucleosome unit
is repeated over and over and creates beads on a string
Te way in which a cell packages its chromatin
during interphase leads to two general forms
Euchromatin:
formed around sites of active genes
Heterochromatin
formed in regions of inactivity
Compare and contrast of Euchromatin and heterochromatin
When handling biological evidence, avoid contamination by not
-touching, sneezing or coughing over evidence
- always use clean gloves and tools
-change gloves between each otem you touch
To collct stains on a surface:
-wet stains: use sterile swabs or gauze
-dry stains: use distilled water ( for rehydration) and sterile swabs, or carefully scrape/cut the stained surface
You can locate invisible stains by:
Black light: excites proteins and metabolites in fluids, making them fluoresce
Alternate light sources: provide a range of wavelengths + filters to enhance faint stains
Biological evidence packaging:
-air dry thoroughly before packaging to prevent mold and DNA degradation
- Use proper packaging materials like paper bags or envelopes
- Clearly label for chain of custody by including case#, item #, date, intitals, signature
-Document every transfer to maintain chain of custody records
Biological evidence transport and storage
-Transport quickly and use secure, contamination-free evidence boxes
- Store dry and cold
-Short term: 4 C
Long term: -20 C or -80C
What is forensic Serology?
detection and identification of biological fluids using laboratory tests
Presumptive tests:
-Simple, quick, low-cost tests that detect the likely presence of body fluids
-use minimal material and preserve DNA integrity for further testing
Confirmatory tests:
- Provide definitive identification of a specific biological material
Designed to avoid cross-reaction with other fluids or species
Antigens
Molecules(often proteins or carbohydrates) on the surface of cells that can trigger an immune response
Antibodies:
-Immunoglobulins
- Proteins produced by B lymphocytes in response to specific antigens
- They bind to antigens, marking them for destruction or neutralization
Antigen-antibody reaction:
-immunological specificity
-essential for forensic serology tests
What are the limitations of blood antigen typing?
-Many people share blood type
- Antigens can degrade over time, especially in adverse environmental conditions
What are immunoassays?
Analytical tests that detect or measure substances( analytes) in a sample using highly specfic antigen-antibody reaction
Immunoassays can be aplied in
Drug screening, species identification, detection of body fluids, and hormone levels
What is the purpose of ELISA?
Uses an enzyme attached to an antibody to produce a measurable signal (color change) when the antigen-antibody reaction occurs
Modern role of serology:
-still crucial for initial screening, species identification, and preliminary drug testing before DNA or more advanced chemical analysis
The advert of DNA profiling (RFLP, STR) along with biotechnology (PCR, MPS):
-Revolutionized forensic individualization by providing far greater discriminatory power, even from degraded samples
VNTRs are
-short DNA sequences repeated consecutively at specific chromosomal locations
- ideal genetic markers for individual identification
- high polymorphic and distributed throughout the genome
-This variation creates different allele sizes
-Molecular scissors that cut DNA at specific sequences
Restriction Enzymes
What is a DNA probe?
-a short, single-stranded DNA fragment that binds to a complementary sequence in the target DNA
- must be labeled to make the target visible
- DNA samples must be denatured to single strands so the probe can hybridize to matching targets
What are the advantages of RFLP?
- high discriminating power: can individualize DNA to a very high degree, especially when multiple VNTR loci are analyzed.
- revolutionized forensic investigations by linking biological evidence directly to individuals
Limitations of RFLP:
-Time-consuming and labor-intensive, with multiple steps, and takes weeks for results
- requires large amounts of highly quality DNA (not suitable for trace samples)
- susceptible to degradation
- Cannot be easily automated
- paved the way for new technologies
(PCR) DNA template:
target DNA to be amplified
(PCR) Primers:
Short, synthetic single-stranded DNA sequences that are complementary to the ends of the target DNA region. They define what gets amplified
(PCR) DNA polymerase:
An enzyme that synthesizes new DNA strands ( Taq polymerase, which is heat-stable)
(PCR) Deoxyribonucleotides dNTPs:
the building blocks for new DNA strands (A,G,T,C)
(PCR) Buffer:
provides optimal conditions (pH, salts) for the reaction
For the visualization, nucleic acids are
stained using intercalating dyes that insert themselves and can be seen under UV or blue light, and the results are qualitative and presence/absence
Limitations of PCR:
-Contamination risk: are highly susceptible to contamination
- Inhibitor: substances in forensic samples can inhibit PCR reaction, leading to false negatives
- Prior sequence knowledge: requires knowledge of target DNA sequence to design primers
- Limited fragment length: PCR is best suited for amplifying relatively short DNA fragments
Short tandem repeats:
-The current gold standard
-has short repeats, which are ideal for PCR