Barcoding lab manual test

two components of DNA barcoding

1. genetic tag or barcode
2. an electronic database capable of providing the identity

DNA barcode definition

short standardized genomic region represented by its constituent nucleotide sequence

what type of DNA is used?

genomic DNA (specific loci)

standard barcode region for animals

650 bp segment of mitochondrial COl gene

standard barcode region for fungi

nuclear ribosomal ITS

standard barcode region for land plants

and nucleotide sequences from two chloroplast genes- rbcL matK

reference DNA barcode

stored in barcode of life data systems reference library

query barcode

unknown sample which is compared to the reference

characteristics of COI reference barcode

must be at least 500 nucleotides in length, less than 1 percent ambiguous base calls, no stop codons, contaminating sequences, insertions or deletions

impact of ethanol (lab 1)

to obtain DNA molecules extruded from cells

* if present during lysis, it will reduce gDNA yield

role of the digestion buffer step 1 (lab 1)

causes the cell membrane to disintegrate

role of proteinase K (lab 1)

responsible for digestion of all the proteins in the cell lysate

role of incubation in DNA extraction

ensures complete lysis of cells in the suspension

Morphological characteristics

Physical characteristics

Conserved region of DNA

Nucleotide sequence that has little to no variation across species

general steps

Collect samples \n Classify species \n Genomic DNA preps \n PCR each sample \n Agarose gel \n Sequence DNA \n Analyze sequence

Steps for DNA barcoding

Digestion \n Isolate DNA \n Purification \n Amplify DNA with PCR \n Gel electrophoresis \n Sequence DNA \n Analyze

Digestion of DNA

Extract genomic DNA from sample \n Mechanically break down membranes in bead tube homogenizer \n Lysis solution

Lysis solution

Digestion \n Buffer that helps to break down the membrane of nuclei \n Contains EDTA

Purifying DNA

Purify DNA from tissue by removing proteins, RNA, and other cellular components before doing PCR

Isolating DNA

Isolate DNA using a spin column based DNA extraction

Nuclease

Enzyme that breaks down DNA or RNA

Direct amplification

Adding untreated sample tissue directly to PCR tube and running the reaction

How was the bacteria sample amplified?

Directly amplified

Why to we process the sample in PCR?

Reaction can sometimes be inhibited by presence of denatured proteins, RNA, lipids, and other cellular materials

If cells do not lyse, DNA will not be released into reaction and there will be less template to amplify

What happens if there is less template to amplify?

Contaminating compounds and reduced levels of template decrease success rate of PCR

Purifying DNA

Purify DNA from tissue by removing proteins, RNA, and other cellular components before doing PCR

Isolating DNA

Isolate DNA using a spin column based DNA extraction

What happens in the spin column?

Purification/isolation

Lysate made from digestion is added to the spin column and centrifuged

Why does DNA bind to the spin column?

Purification/isolation

There is an ionic interaction between DNA and the column's membrane in the presence of salt

What is added to the spin column with DNA?

Purification

Wash buffer containing ethanol to remove any other contaminates including proteins, lipids, salts, or RNA

DNA is not highly soluble in ethanol , so DNA remains bound to column

Why is water added to the spin column?

Isolation

DNA is very water soluble

Water is pulled through the column contains isolated genomic DNA

Why do we have to run PCR on the DNA we obtained from purification?

The amount of DNA is too little to directly sequence

There are numerous DNA fragments that we do not want to sequence that may interfere with reactions

PCR to amplify the specific barcode fragment of DNA that we want to sequence

Constituents required in replication reaction

DNA polymerase to synthesize new DNA

Deoxynucleotide triphosphates (dNTPS) for incorporation (dATP, dGTP, dCTP, dTTP)

Short oligonucleotides that serve as primers to which DNA polymerase adds the dNTPs to extend the DNA strand

DNA for a template

Why is Taq polymerase used?

Enzymes will not degrade at 95 C

Optimal temperature is 72 C

Steps of PCR

Denaturation

Primer Annealing

Synthesis

Denaturation of DNA

PCR

Purified DNA is placed in microcentrifuge tube and heated

95 C

Heating disrupts hydrogen bonds holding the strands of DNA double helix together

Double-stranded DNA denatures into single-stranded DNA

Primer Annealing of DNA

PCR

Taq polymerase can only synthesize DNA on a naked single-stranded DNA, but can only extend from a primer

Use forward and reverse primers

50 C

Two primers anneal with DNA

Synthesis of DNA

PCR

Taq polymerase to synthesize DNA from two primers

72 C

DNA primers

Use forward and reverse primers

Hybridize on either end of the region we want amplified

Will only amplify the DNA between primers

Designed to anneal to highly conserved regions od DNA within the barcode gene

Segments of DNA between primers have enough nucleotide sequence variation across organisms to allow for species identification

Barcode markers

COI gene (animal)

rbcL gene (plant)

16S gene (bacteria)

COI gene

Codes of Cytochrome C Oxidase

Highly conserved gene that is found in mitochondria and is important for respiration

Animal samples

rbcL gene

Codes for ribulose-biphosphate carboxlyase (RuBisCo)

Found in chloroplast genome

Involved in carbon fixation in one of the first steps in photosynthesis

Provides a DNA barcode marker to specifically identify plants and enables scientists to distinguish the plant from other organisms that may be living in or on the plant

Plant samples

16S rRNA gene

Codes for 16S ribosomal RNA that is part of the bacteria ribosome complex

Does not code for a protein

PCR master mixes

DNA polymerase

dNTPs

Forward and reverse primers

Water

Why is a master mix used in PCR?

Reduces pipetting errors and time spent running reaction

Gel electrophoresis

Determine if PCR was successful and the size of resulting DNA fragments

Standard method used to separate, identify, and purify DNA fragments

Why do DNA fragments move through the gel?

Phosphate groups on DNA backbone confer a net negative charge on molecule

Solution of DNA placed into an electric field, DNA molecules migrate toward positively charged electrode

Agarose gel

Porous matrix

When DNA moves through gel, it must "snake" around agarose, fitting through the pores

How does gel electrophoresis separate DNA by size?

Smaller fragments of DNA migrate more easily and faster through pores than larger fragments

What is added to the DNA before loading it into the gel?

Blue gel loading dye

What does the blue gel loading dye consist of?

Glycerol: DNA sample to sink to the bottom of the well

Xylene cyanol and bromophenol blue: tracking dyes

Why is ethidium bromide added to the gel?

DNA is not normally visible to the eye

EtBr binds to the DNA and shines bright red when exposed to UV light

What does the presence of the ladder in the gel indicate?

Gel was made properly, electrophoresed in the correct direction, and DNA was stained by ethidium bromide