Anatomy and Physiology 12: DNA and Gene Expression Unit PDF

**Anatomy and Physiology 12 : DNA and Gene Expression Unit** **Clonging/Sequencing/Analyzing DNA** Who here likes crime shows? Paternity testing drama? Do you know what types of methods they use to catch suspects? -usually we hear lots about PCR -- polymerase chain reaction History of DNA sequ...

**Anatomy and Physiology 12 : DNA and Gene Expression Unit** **Clonging/Sequencing/Analyzing DNA** Who here likes crime shows? Paternity testing drama? Do you know what types of methods they use to catch suspects? -usually we hear lots about PCR -- polymerase chain reaction History of DNA sequencing and cloning -Kary Mullis invented PCR in 1983 (relatively recent discovery) and won a nobel prize-- revolutionary because we weren't able to acquire large quantities of specific sequences of DNA prior -- enabled scientists to identify genes and connect them to diseases, do forensic/paternity testing -my old biology professor used to talk about Mullis being strange -- believes climate change isn't related to human activity, HIV doesn't cause AIDS, and used to cause personal conflicts with coworkers Old cloning methods use bacteria -- see page 129 in your textbook -- we will get there soon when we would do that **Modern Cloning - Polymerase Chain Reaction (PCR**) -- make millions of copies of a specific DNA sequence Youtube: https://www.youtube.com/watch?v=a5jmdh9AnS4&t=273s Need 3 things: Taq polymerase -- a heat stable version of DNA polymerase 2 Oligonucleotide (short section of nucleotides) Primers -- act like pieces of bread that sandwich the DNA sequence of interest Lots of A,T,G,C nucleotides for building DNA copies **PCR 3 basic Steps:** 1. [Denature] the DNA helix into two separate strands using high heat 2. Let the two oligonucleotide primers [anneal] 3. Taq polymerase extends off the primers until it reaches the end of the DNA strand Benefits of PCR? -- only need a really small sample, and it is entirely what you want **How can we use PCR for forensics?** - Recall that we are [diploid] organisms -- we have 23 chromosomes and there are 2 copies of each - Our DNA has some [conserved] (found in everyone) sections of DNA -- one example of these are [short tandem repeats] (STR's) -- short sequences of DNA that repeat (1-6 base pairs long) - However, there are slight variations of the number of repeats people have - We can use PCR to amplify these sections, then run the DNA using gel electrophoresis to separate out the different size pieces -- will appear as dark bands on the gel -gel electrophoresis uses electrical current to push DNA through small pores in the gel -- larger fragments get stuck and travel less distance - How many bands would you see if you were homozygous? (1) Heterozygous? (2)  In practice, we would sample multiple STR locations, so that you would see a bunch of bands -- see electrophoresis gel below **Now we know how to clone DNA -- how do we determine which nucleotide is where in a sequence?** [Sanger Sequencing] or **[Chain Termination]** Sequencing https://www.youtube.com/watch?v=dVRB4CaLizc - Follows the same general process as PCR, except that fluorescently labelled dideoxynucleotides are included (different label for each base) -these nucleotides are missing the 3' oxygen needed to form additional phosphodiester bonds -- DNA synthesis stops -eventually you will have a whole bunch of labelled DNA strands differing in length by 1 nucleotide - Capillary gel electrophoresis separates the fragments by size -- computer detector reads fluorescence passing by -- tells us the sequence - Many other next generation methods on the way **Human Genome Project was enabled by these recent technological developments** - 13 year project -- started in 1990 and ended in 2003 - Our entire genome has been sequenced -- how does this help us? -see single nucleotide polymorphisms (SNP's) between individuals -- related to disease? -we have 25,000 genes and a ton of non-coding DNA -- what is its purpose?