Biotechnology – Making More DNA

1. Biotechnology – Making More DNA

2. Agenda • Chromosomes and Genes • DNA Replication - transcription • RNA Replication – translation • “Natural Mutations” • Where biotechnology comes in • Where GMO’s come in

3. Chromosomes • What are they? • Where are they? • What’s in it for me?

4. Chromosome – What? • Twisted strand of bunched up nitrogen compounds • Nitrogen compounds – aka: nucleotides • Nucleotides match up to make a pair • This pair gets twisted • The twist bunches up • Forms an ‘X’ shape • You know it as “DNA”

6. DNA – What? • Chromosomes come in pairs, there are two identical chromosomes with the same nucleotides at all times. • The number of pairs it takes to have ALL the genetic information depends on how complex of an organism you are. Or it’s just random. • Your DNA is not found on just one chromosome!

8. Chromosome – Where? • Found inside each nucleus • When cell divides (you grow, need to replace a cell) the DNA replicates inside the nucleus • Other stuff happens in the cell, but we’ll get to that later

9. What’s in it for me? • Human DNA – 22 pairs of chromosomes + 1 pair of sex chromosomes = 23 pairs • Many ‘diseases’ are due to genetic ‘oopses’ = down syndrome, spinabifida, Kleinfelter’s disease • Your DNA is constantly changing too!

11. DNA Replication - Transcription • How it works • Why it’s called ‘transcription’ • How it can get all wrong (intro)

12. Time to Replicate • Cell tells nucleus: replicate • DNA/chromosomes come out of solution and solidify • Chromosomes line up along the middle of the nucleus • The chromosomes are ripped apart and taken to the poles (only half of a chromosome will be on each half of the nucleus) • The nucleus (and the whole cell) will ‘bud’ into two cells, each will have ALL the genes, but only as a ‘half’

14. More to Replication • Now that there is DNA in each new cell, it’s time to fully replicate! • Enzymes (proteins) un-zip the double helix and begin replicating in 2 directions! • One strand will replicate from beginning to end: leading strand • One strand will replicate bits and pieces in the opposite direction: lagging strand

16. Transcription for Genes • Remember: DNA has ALL genetic information. Let’s say it’s time for us to grow again. We need our DNA to tell us to grow: genes are ‘turned on’ and start replicating DNA to make stuff happen. • This is called “transcription” (think ‘prescription’ for things) • As the DNA is being un-zipped and copied, mRNA is making RNA (DNA that can be read by ribosomes) instead of DNA. • RNA will leave the nucleus and go into the cell (DNA stays ONLY in the cell!)

18. Where it can all go Wrong • While the enzymes are reading DNA, making more DNA or making RNA, bad things can happen. • Each nucleotide in DNA is called a ‘base pair’ • The entire human genome has 3.2 billion bp 3,200,000,000 base pairs • There are about 23,000 protein-codes to miscopy

19. Bad Things Include: • Deletions • Duplications • Inversions • Translocation • Insertion • Transitions • Transversions • Tautomeric Shifts • Simple Sequence Repeats • Spontaneous Lesions • And many more!

20. RNA Translation • What is it? • Why is this super important?

21. RNA Translation – What? • RNA translation happens outside of the nucleus (in the cell) • RNA is the ‘working’ DNA – it tells proteins to form, perform, get torn apart, start and stop other proteins, etc • Different set of nitrogen compounds (hence the R instead of D)

22. RNA – How? • Ribosomes come and read the strip of information • The information tells them to bring amino acids (one chunk of a protein) • These AA’s build into a chain • This chain gets twisted and bunched • Eventually will form a protein

23. Insulin for Instance • Your body needs to build insulin (protein) after you eat • Insulin breaks down sugars • If your body doesn’t have accurate DNA to make accurate RNA, your body can’t make insulin • Without insulin, the sugars in your blood will rot the vessel walls and you die from hemorrhaging • This genetic disease is called ‘diabetes’

24. Mutations - Naturally • Point Mutations – these are small and only affect one or 2 genes, or even nucleotides • Transposons – ‘jumping genes’ that move around in the genome (happens in meiosis) • Deletions – large pieces of DNA missing/never got copied • Duplications – extra repeats (think Down Syndrome) • Rearrangement – when a piece breaks off and • Inversion – gets attached backwards • Translocation – attached in a new location

25. Mutations - Naturally • Single Nucleotide Polymorphisims (small changes) • Insertion – extra base (extra A) • Deletion – missing base (took out an A) • Transition – (A-G or C-T) • Transversion – (A-T or G-C)

26. Mutations – Not Natural • Tautomeric Shifts – chemical change in base structure • Simple Sequence Repeats – AT= ATATATATATATA • Spontaneous Lesions – don’t know reason for change • Base Analogs – chemicals alter A – T, C – G • UV Radiation – morphs into unreadable shape

27. Making more DNA – Science! • PCR – DNA photo copier • ONLY COPIES DNA! • Repeat up to 30-40x!

28. Making RNA – Science! • RT-PCR – RNA photo copier! • Copies into DNA! • Reverse enzyme reads the RNA backwards and then makes the matching DNA

29. Making Read-Outs of DNA • Need to cut DNA down to size • We use ‘restriction enzymes’ • They ‘cut’ the DNA at certain locations in the sequence • BAM HI Cuts at G^GATCC • EcoRI cuts at G^AATTC • HaeIII cuts at GG^CC

30. Electrophoresis • The number of cuts made makes DNA into shorter (smaller) chunks • Run on a sponge, the big pieces get caught at the ‘top’ while the smaller pieces can float down to the bottom • Agarose gel electrophoresis!

32. Uses? • Allowed us to map the human genome over several thousand times