DNA Technology

a Dr. Production

DNA Coiling:

Replication: DNADNA• Occurs during S phase

of mitosis in reproducing cells only

• DNA template is copied complimentarily and semi conservatively

• Multiple oris (in eukaryotes) and replication forks form in both directions, producing a leading and lagging strand as DNA is elongated to the 3’ end

Complimentary & Antiparallel

• DNA end with the phosphate attached to the #5 carbon = 5’ end

• DNA end with the OH group attached to the #3 carbon is the 3’end

• Synthesis of a n.a. chain always proceeds 5’ to 3’

Okazaki Fragments

• Fragments of DNA 1000-2000bp long

• Produced on the lagging strand

• Must be joined with ligase

Replication:

DNA Replication Fork & Nucleotide Addition Videos

:

Enzymes• DNA polymerase1:exonuclease activity, 2: DNA repair,

3:primary replication enzyme)• Ligasejoins nucleic acid pieces• Primase joins RNA primer to DNA strand• Helicaseunwinds the DNA• Nuclease cuts DNA• Endonuclease cuts DNA internally vs at the ends• Telomerasecatalyzes lengthening of telomeres• Topoisomerase lt rs t a e he degree of

supercoiling nucleic acids but cutting and rejoining strands

Mistakes/Mutations

• Mutation any permanent alteration in the sequence of DNA bases

• Point mutations can result in frameshifts in the reading frame

• Chromosomal mutations can result in translocations

Chromosomal Mutations

Base-pairing (substitution/ deletion-addition) Mutations

Mistakes/Mutations

• Silent mutation change in nt sequence, but no change in product

• Nonsense mutation insertion of stop codon into sequence

• Missense mutation swapping of aa (sickle cell anemia, PKU)

Types of DNA

Coding DNA

Less than 5% of the human genome actually codes for genes

Simple Sequence DNA

20-30% human DNA

Short sequence of 5-10 bp repeat.

Half of the DNA in a species of crab has ATATATATA

Fruit fly has ACAAACT 12 million times

Intermediate-Repeat

150-300bp scattered throughout genome, some have known functions and code for histones and rRNA

Huntington’s disease CAG repeats 6-35/38-180bp (also various dystrophies)

Junk DNA ?More than 95 percent of all DNA, was called "Junk DNA" by molecular biologists, because they were unable to ascribe any function to it. However that category is becoming smaller as scientists identify non-coding DNA as introns, transposable elements, pseudogenes, VNTRs (variable number tandem repeats and “junk”.

Chromosome #11 Flyover

Extraneous DNA

proviruswhen the DNA of a virus incorporates its self into an eukaryotic cell. Can move around in the genome

reverse transcriptase enzyme that allows RNA strand to be a template of DNA

transposons “jumping genes”

Where did it come from?One hypothesis about the junk is that these chromosomal

regions are trash heaps of defunct genes, sometimes known as pseudogenes, which have been cast aside and fragmented during evolution.

Evidence for a related hypothesis suggests that the junk represents the accumulated DNA of failed viruses.

Yet another hypothesis is that the junk DNA provides a reservoir of sequences from which potentially advantageous new genes can emerge. (Junk vs trash)

As with most things in science, time and technology will tell…

Types of RNA• mRNA “messenger RNA” Transcribe DNA to RNA• rRNA “ribosomal RNA’ used in the building of ribosomes• tRNA “transfer RNA” carry a.a. to ribosome during translation• snRNA “small nuclear RNA” Mediate the processing of DNA

transcripts for mRNA, rRNA, and tRNA • snoRNA “small nucleolar RNA”RNAs within the nucleolus have

several functions • miRNA“micro RNA” tiny (~22 nts) RNA molecules that appear to

regulate the expression of mRNA• XIST RNA. inactivate one of the two X chromosomes in female

vertebrates. • gRNA “guide RNA” found in mitochondrial DNA• sl RNA “splice leader RNA” 5’mRNA cap• hnRNA “heterogenous nuclear RNA” nonspliced or immature RNA

Isolating DNARestriction enzymes/

endonucleases cut at specific palindromic sequences

5’ G/AATT C 3’3’ C TTAA/G 5’

cleavage leaves “sticky ends” G AATTCCTTAA G

Steps in Cloning a GeneDNA Restriction Step by Step

Recombination• Recombinationprocess in which one or

more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence

• In eukaryotes, usually occurs as the result of crossing-over during meiosis

• In prokaryotes, usually occurs as the result of plasmid DNA– Construction of a Plasmid Vector

– Mechanism of Recombination

– DNA Transformation

RFLP Analysis• Restriction Fragments. Those are the fragments of DNA that were cut

by restriction enzymes. • Length, and refers to the length of the restriction fragments. • Polymorphism, a Greek term that literally means "many shapes". The

lengths of some of the restriction fragments differ greatly between individuals, thus there are many shapes, or lengths, of DNA possible.

• Molecular biologists have identified regions of the human genome where restriction fragment lengths are highly variable between individuals. These regions are called RFLP markers.

VNTR Tutorial

Electrophoresis

• Move DNA through a matrix with a sieving effect

• Agarose gels for DNA• PAGE for proteins• Single digest uses 1

enzyme• Double digest produces 2

restriction patternsInteractive Animation, Simplified

Blotting

• Goes from the gel to filter paper to make and autoradiograph

• Southern blot: uses RNA to probe DNA strands (SB2)

• Northern blot: uses DNA to probe RNA strands

• Western blot: “immunoblotting” uses pp-antibody binding reaction

Hybridization

• These hybridization reactions can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe.

• used to find relatedness in n.a.

Polymerase Chain Reaction (PCR)

PCR2 PCR3 PCR4 PCR Rap PCR Song

Transcription: DNAmRNA

• Occurs continually throughout the cell cycle

• Occurs in the nucleus as messenger RNA copies DNA

• cDNA

• Operon=promoter + operator + TFs• Promoter region, upstream of gene, signals start of

transcription• Transcription factors mediate binding of RNA polymerase• Operon is controlled by regulatory genes such as repressor

genes

Control in Prokaryotes: http://trc.ucdavis.edu/biosci10v/bis10v/week6/08lacoperon.html

Transcription Initiation Complex

• Non-coding regions (introns) of mRNA are spliced out of the mRNA strand before leaving the nucleus

• 5’cap is methylated and 3’tail is polyadenylated to indicate orientation at the ribosome

Splicing of introns & exons

Translation: mRNAprotein• Aamino acyl (initiation with Met.)• Ppeptide bond (elongation)• Eexit (termination with UAG, UAA, UGA)

Regulatory Proteins

• Lac Operon

• Lac Operon Induction

• Regulatory Proteins: Repression

Signal Transduction

• Signal Transduction Pathway

• My Dog is Broken (A Case Study for Cell Signaling)

GMOs

• Classical vs Transgenic Breeding

• Harvest of Fear (What about this fish?)

• Babies by Design?

• Transgenic Mouse

Gene processing

• BioStudio Animations: http://www.biostudio.com/a_sitemap.htm

• Biology Animations: http://science.nhmccd.edu/biol/ap1int.htm

• Restriction Mapping Tutorial:

• DNA Replication:

• BioZone BioLinks: http://www.biozone.co.nz/CELL_BIOL_AND_BIOCHEM.html

• Lac Operon Animation:

• Lac Operon Induction:

• Biointeractive Animations:

• Animated Tutorials: Molecular Biology:

• Processing of Gene Information Animation Prokaryotes vs Eukaryotes:

• Learner’s TV:

• Fundamentals of Molecular Diagnostic Links:

• Gene Expression

• DNA Technology Quiz

Resources