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Unformatted text preview: Bio 106 Fall 2009 Professor Owen 10/17/2009 Telomeres
What is the problem with replication at the end of the chromosome? Think about the end of the chromosome, especially especially the lagging strand: Can it be replicated normally? Can you sketch its replication? A. No room on lagging strand to make the primer B. No room on leading strand to No room on leading strand to make make the primer C. No problem D. No E. N Telomeres Telomeres Think about the end of the chromosome, especially especially the lagging strand: Can it be replicated normally? Can you sketch its replication? A problem: Since DNA polymerase only works works in 5’ 3’ direction, it runs out of room at the end of the chromosome. The enzyme telomerase lays down an extra length to the 3’ template end (a series of repeating sequences). ECB3 6-18 ECB3 6-18 2009 Nobel Prize in Physiology or Medicine
Carol Greider, Elizabeth Blackburn and Jack Greider, Szostak A Single BP Change (review) Sickle cell anemia is caused by a single base-pair inversion (genetic basis).
ECB2 5-11, 6-19, 10-13 Cells17-PCR & Transcription 1 Bio 106 Fall 2009 Professor Owen 10/17/2009 DNA Can Change Over Time
Why does cancer incidence increase with age? ith
Over time we accumulate damage to our DNA from environmental and chemical toxins, and even sunlight.
ECB3 6-20 UV Damage UV light In skin cells, for example, two adjacent thymine bases can become covalently attached. Cooper 6.18 Nucleotide-excision Repair
Damaged DNA is: • recognized • cleaved • resynthesized • resealed DNA polymerases from different species have different characteristics. We use this to our advantage in the laboratory. Taq ECB3 6-20 Taq polymerase was isolated from the bacteria Thermus aquaticus DNA Melting
Heat causes the DNA double helix to come apart into single-stranded DNA Once this DNA. Once this starts (the Hbonds are breaking), it happens quickly. In a DNA melting experiment (Tm), what would you expect to see from DNA with a higher ratio of GC than AT? The melting temperature (Tm) depends on the concentration of G C to A T. A. B. C. D. E. no difference higher Tm lower Tm no Tm Tm > GC>AT Cells17-PCR & Transcription 2 Bio 106 Fall 2009 Professor Owen 10/17/2009 PCR Reaction ECB3 video 10.1 Cooper 4.23 Amplification of DNA by PCR
A good animation is available on the Cooper website (4.9) By providing a known set of primers, PCR exponentially multiplies the number of copies of a gene of interest. PCR Primers
• PCR primers are short (15 – 20 bases) pieces of DNA that are specifically designed to match targeted areas of a gene (The cell uses primers of ____ gene. (The cell uses primers of RNA ). • Two unique primers are typically used in a PCR reaction. • They also provide the 3’ end for Taq PCR
As the number of cycles continue, the sequence of interest exponentially increases. increases.
ECB3 10-15, 16 Why do we need to use a special DNA polymerase for PCR? PCR
• PCR is used to amplify a DNA sequence • DNA primers (one for each strand) determine the region to be amplified • Requires the use of Taq has ability to withstand high heat needed to separate the DNA strands
– Most proteins denature at high temps A.lower A. lower cost B.sounds B. sounds better on an NSF grant C.heat C.heat denatures proteins; Taq Taq can function in the heat D.cold D.cold freezes proteins; Taq can Taq function in the cold Cells17-PCR & Transcription 3 Bio 106 Fall 2009 Professor Owen 10/17/2009 During PCR, what is the function of the primers? How are PCR primers different than those used during the cell’s DNA replication? A.To A. To provide Taq a 5’-end 5’B.To B. To provide Taq a 3’-end 3’C. To provide RNA polymerase a 5’5’-end D. To open the dsDNA A. Not – PCR primers are identical B. PCR primers are labeled with GFP C. PCR primers are ribose-based riboseD. PCR primers are deoxyribosedeoxyribosebased DNA Activities
• Replication= making more DNA helices • Transcription= making an RNA copy of DNA
(Translation = turning DNA code into linked chain of amino acids) Transcription: DNA to mRNA ECB2 7-1 How is RNA Different Than DNA? How is RNA Different Than DNA? Ribose sugar has an hydroxyl group (OH) on Deoxyribose is a ribose that is “missing” one
oxygen (2’ carbon).
ECB3 7-3 both the 2’ and 3’ carbons. RNA contains uracil (U) in place of thymine (T). U basepairs with adenine (A). ECB3 7-3, 4 Cells17-PCR & Transcription 4 Bio 106 Fall 2009 Professor Owen 10/17/2009 RNA
RNA chains have the same sugar-phosphate backbone as DNA (with a different sugar subunit).
note U and ribose Transcription Copies One Strand of DNA into an RNA
The strand opposite the template strand is sometimes called the coding strand because its sequence is equivalent to the RNA product.
ECB2 7-6 Thus, RNA also has a 5’ phosphate end and a 3’ hydroxyl end. ECB3 7-3 RNA Polymerase Copies One Strand of DNA into an RNA
To To which end of the growing RNA would you expect the RNA polymerase enzyme? A. 5’ B. 3’ C. NH3+ D. COOCOOE. inorganic phosphate RNA polymerase unwinds the DNA, adds RNA nucleotides using the DNA template, then allows DNA to rewind into a helix. ECB3 7-6, 7 RNA polymerase reads the template strand in a 3’ 5’ direction, making RNA in 5’ 3’ direction.
movie 7.2 Transcription Types of RNA Transcription can be seen in the TEM. The image shows that many RNA polymerase enzymes can simultaneously transcribe a gene, and more than one gene can be transcribed at a time.
ECB2 7-8 There are different types of RNA, each with specific functions. Three different RNA polymerase enzymes synthesize these RNAs.
ECB3 Cells17-PCR & Transcription 5 ...
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