Lab 7 - DNA

DNA in eukaryotes

nucleus

DNA in prokaryotes

nucleoid region

DNA

“blueprint for life”

contains information → makes proteins → control cellular processes (metabolism, enzyme reactions, photosynthesis, muscle action, brain activity)

DNA structure

double-helix composed of four nucleotides

nucleotide structure

1) nitrogen containing base - unique to specific nucleotide

2) deoxyribose sugar

3) phosphate group

what does ATGC stand for?

adenine, thymine, guanine, cytosine

complementary base pairing

nucleotide pairs through hydrogen bonding

nucleotide A pairs with

T

nucleotide G pairs with

C

base pair

two hydrogen-bonded nucleotides

sugar-phosphate backbone

nucleotides of each strand covalently linked by sugar phosphate groups

eukaryote chromosomes

multiple linear chromosomes

prokaryote chromosomes

single circular chromosomes

DNA function in euk and pro cells

same

genes

nucleotides with chromosomal DNA info

species → chromosomes → genes → position

same

alleles

different forms of the same gene → different types of same protein → not identical in same species

polymorphisms

characteristics due to 0.1% differences within nucleotide sequence of genome

polymorphism function

DNA that differs in length between individuals, used as unique identifiers analyzed through DNA fingerprinting

Variable numbers of tandem repeats (VNTRs)

minisatellites, DNA sequences between 20 to 100bp in length, repeated multiple times

highly variable: different individuals = different numbers of repeats

short tandem repeats (STRs)

microsatellites, DNA sequences between 3 to 10bp in length, repeated multiple times

highly variable: different individuals = different numbers of repeats

restriction enzymes

restriction endonucleases, cut DNA molecules, have cut-site specificity

recognition sequence

where a cut is made only on a particular DNA sequence

restriction fragment length polymorphism (RFLP) analysis

basis of the specific cut-site of restriction enzymes for producing a DNA fingerprint

knowing recognition sequence + sequence of a DNA molecule =

know where to make a cut and the number and size of the DNA fragments

genomes + VNTRs + STRs =

cuts of different fragment lengths by a specific restriction enzyme

DNA gel electrophoresis

separate DNA fragments of different lengths

DNA molecules are negative, attracting

positive

gel electrophoresis chamber

water-tight apparatus, anode (+) + cathode (-) + agarose gel

agarose gel

thin slab of semi-solid gelatin matrix that has tiny pores, DNA fragments mmove through

electrophoresis buffer

ions inside allow it to carry electrical current

loading wells

empty spaces on one end into which DNA samples are loaded

smaller DNA fragment =

faster migration and separation of DNA fragments by size

band

millions of DNA fragments of equal length collecting in same location

DNA gel electrophoresis process

1) gel placed into chamber with wells closest to cathode

2) chamber filled with electrophoresis buffer

3) DNA samples loaded into wells

4) chamber connects to power supply and voltage is applied

5) DNA fragments migrate to positive end and separate by size

6) complete: small DNA fragments with anodes, large DNA fragments with cathodes, same size in same location

DNA fingerprint

created by nucleotide polymorphisms, unique banding pattern from a DNA sample based on size and number of DNA fragments

little DNA/cells needed to

produce DNA fingerprint using STRs

standard curve

determines DNA fragment length using a DNA ladder containing DNA fragments of known lengths

load DNA samples in one lane + load DNA ladder sample in separate lane + electrophoresis process completion =

DNA fragment length standard curve → determines sample fragment length

solid lines between nucleotides represent

covalent bonds

dotted lines between nucleotides on different strands represent

hydrogen bonds

white rectangles represent

loading wells

black rectangles represent

bands

purpose of the detergent in lysis buffer

break down cell and nuclear membrane by dissolving lipids, allowing DNA to be released from the cells

purpose of salt in lysis buffer

separates DNA from proteins and helps DNA molecules clump together so they can precipitate out of the solution and be easily seen

octoploid genome

eights sets of chromosomes

diploid genome

two sets of chromosomes

strawberries are octoploid so

each cell contains a larger amount of DNA that makes it easier to extract and see the DNA as we demonstrate DNA isolation