Lab 4: Forensic Biology and Bioinformatics I

gene theory

the biological principle that traits are passed from parents to offspring through the transmission of genes

genome

the complete set of DNA in an organism

chromosome

thread-like structure located inside the nucleus of a cell, composed of DNA tightly coiled around proteins called histones, which carries the genetic information of an organism

DNA

the molecule that carries genetic information for the development and functioning of an organism

gene

a segment of DNA that contains instructions for making a specific protein

transcription

DNA→ RNA

translation

RNA → protein

what structure is DNA?

double helix structure

what is DNA composed of?

nucleotides

4 building blocks of DNA

ATCG, and U in RNA

the two strand in DNA molecules are:

• complementary

• antiparallel

coding DNA

• encodes for protein-coding genes

• consists of the coding region of protein-coding genes aka exons

• only 1% of human genome

noncoding DNA

• 99% of the human genome

• does not encode for protein-coding genes

STR

short-tandem repeats

where are STRs found

in the non-coding region of DNA

what is used to differentiate one person from another?

STRs

what percentage of people share the same DNA profile at any one STR site

5-20%

how many STR regions are used in DNA profiling?

15

Assume that the odds of two people sharing STR patterns at any given STR region is 5% (0.05).

Given this, the probability of two people with matching STR patterns at multiple regions are as follows:

matching at 3 regions: 0.05 × 0.05 × 0.05

what amplifies the STR regions from each DNA sample?

polymerase chain reaction

taq polymerase

a specialized DNA polymerase, used in this reaction because of its ability to survive in high-temperature environments

what does the denaturation of DNA result in?

the hydrogen bonds between complementary base pairs being broken yielding only single stranded DNA

primer

the annealing/recombining of short pieces of DNA, specific to the sense or antisense strands bind via hydrogen bonds

elongation using polymerase

beings by adding free dNTPs to the ends of the primer one at a time in the 5’ to 3’ direction to make double stranded DNA

DNA polymerase

taq polymerase is a thermostable enzyme isolated from the bacterium Thermus aquaticus. can withstand temperatures greater than 90 C

amplicon

short fragment trying to amplify

how does the amplicon form?

when the polymerase extends from the forward primer on a strand that was generated by amplification from the reverse primer or vice verse

deoxyribose nucleoside triphosphates or dNTPs

will comprise the base pairs in the growing strands

reaction buffer

maintains pH and contains important ions like manganese, magnesium, and potassium: stabilizes the reaction and provides important cofactors to the polymerase enzyme

a solvent PCR grade water

free of ions that can inhibit the reaction

chelex resin

absorbs ions that inhibit the function of Taq polymerase

thermocycler

fine temperature control: ability to hold a precisely set temperature with little fluctuation

master mix contains:

• buffer

• loading dye

• deoxynucleotides

• three pairs of primers

• taq polymerase

• water

what does the thermocycler do?

amplifies the STRs in DNA