Forensic Biology Lab Operations Final

DNA Profiling, PCR, RFLPs

DNA profiling

technique used by scientists to distinguish between individuals of the same species using only samples of their DNA

Stage 1 of DNA Profiling

cells are broken down to release DNA

restriction enzymes

proteins/enzymes that break the molecules of DNA into small fragments (“cuts” the DNA at sites)

Stage 2 of DNA Profiling

DNA is cut into fragments using restriction enzymes

restriction fragments

sections of DNA that are cut out

Stage 3 of DNA Profiling

fragments are separated based on band size using gel electrophoresis

T/F: DNA is negatively charged so it will move towards the positive end of gel

true

T/F: short bands move slower than longer bands

false

gel electrophoresis

process where samples of DNA are placed into gel wells and through electrical currents, separate themselves based on their different sizes

uses of DNA Profiling

• solve crimes

• solve medical problems

• establish paternity

genetic fingerprint

the pattern of bands in gel electrophoresis

PCR

common laboratory technique used to make many copies of a particular region of DNA in vitro; goal is to amplify enough DNA to be analyzed

Taq polymerase

isolated from heat-tolerant bacterium (Thermus aquaticus); makes new strands of DNA using existing strands as templates during Extension phase of PCR

PCR primer

short sequence of nucleotides that provide a starting point for DNA synthesis during Annealing phase of PCR

Denaturation

(96 C) heat the reaction strongly to separate the DNA strands; creates single stranded template

Annealing

(55-65 C) cool reaction so the primers can bind to their complimentary sequences on the single stranded DNA template

Extension

(72 C) raise the temperature so Taq polymerase extends the primers, synthesizing new strands of DNA

how many times is the PCR cycle repeated?

25-30 times

PCR requirements

1. sample - template

2. primers

3. buffer

4. Taq polymerase

5. Deoxynucleotide Triphosphates

thermocycler

instrument programmed to change samples rapidly from one set temperature to another

T/F: a large amount of DNA is needed for PCR analysis

false

degraded DNA can be used in PCR so long as the sample is ______ enough

large

applications of PCR

• PCR cloning - cloning of genes

• genetic diagnosis of mutations

• paternity testing

• Reverse Transcription

• forensic analysis of crime scene

• industrial quality control; water quality testing

• rapid detection of potential biological threat agents

• real-time field diagnosis of viruses

primers will:

flank the region of interest, anneal to opposite strands of template, prime toward the region between them

the buffer contributes to what?

correct folding of enzyme and optimum activity of enzyme

T/F: the denaturation temperature must be high enough to overcome the attractive energy of H-bonds between bases of template

true

what determines the optimum annealing temperature?

1. the length of the primer

2. the percentage of Guanine-Cytosine bonds

3. the quantity of salt

the longer the primer. . .

the more Hydrogen bonds, which requires higher optimum annealing temperature

the more Guanine-Cytosine. . .

the higher the optimum annealing temperature

T/F: G-C have only 2 hydrogen bonds while A-T have 3 hydrogen bonds

false

the higher quantity of salt. . .

the more positive counterions present, and the less negative charge of DNA

Tm

temperature at which 50% of the possible correct primer/template complexes are unformed

annealing temperature for PCR is often set at what?

5 C below the Tm

the longer the expected produced, the ________ the extension time required

longer

what are the controls in PCR?

• known positive (should produce a product)

• size markers

what do known positive controls tell you?

that the reaction components are working and/or what your product should look like

should minimize contamination of:

pipettors, supplies, reagents, and work area

primer dimers

short, unintended DNA fragments formed in PCR when primers anneal to themselves or each other and are extended by polymerase

causes of primer dimers

• primer excess too great

• insufficient target template

• too many cycles

• annealing temperature too low

• primers too short

introns

non-coding region of DNA base pairs

out of 3 billion DNA base pairs, about ___ are coding proteins

5%

Alu elements

mostly existing in non-coding regions, 300 bp DNA element in the human genome

T/F: each PCR cycle doubles the amount of the target DNA in less than five minutes

true

T/F: DNA fingerprinting can only exclude a suspect, and cannot provide positive identification

false

child’s DNA

composite of its parents DNA; bands present in the child’s DNA must be found in either the father’s or mother’s fingerprint

Restriction enzymes are ____________ which catalyze the cleavage of phosphodiester bonds

endonucleases

what causes differences in base sequences?

mutations and deletions

restriction fragment length polymorphisms (RFLP)

technique of DNA typing using varying lengths of DNA patterns unique to the individual

Variable Number of Tandem Repeats (VNTR)

RFLP occurring in non-coding region of DNA; segments of DNA contain sequences of 2-40 bases in length and repeat a specific amount of times in an individual

what causes RFLPs?

variations in length of a given segment of genomic DNA between two restriction enzyme recognition sites among individuals of the same species

if RFLP patterns match, it is beyond a reasonable doubt that the suspect was:

present at the crime scene

T/F: DNA fingerprinting analysis includes forensic enzymology, ELISA, and forensic toxicology

false

in forensic cases, DNA samples can be extracted and purified from biological samples, NOT including what?

urine

T/F: DNA evidence is most powerful when used to demonstrate that an individual is NOT the source of biological evidence in a criminal investigation

true

T/F: DNA is high polymorphic, therefore two individuals have the same pattern of restriction enzyme recognition sites in their DNA

false