Pre-lab

Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase.

Gene transfer involving plasmids are usually used to alter the circular DNA molecules called plasmids that are usually found in plasmids. 

Stage 1: Obtaining a gene to transfer

-Restriction enzymes are put in the same container as the DNA strands

-During this stage, restriction enzymes are used to cut the targeted gene that is to be transferred from the DNA of an organism to the plasmid and create 'sticky ends'

Stage 2: Preparing a vector for the transferred gene

-Restriction enzymes are also put in the same container as the plasmids

-The same restriction enzymes that had cut the targeted gene from the DNA strand is used to create a cut in the plasmid, creating 'sticky ends' complementary to the DNA fragments from an organism

Stage 3: Recombinant DNA

-DNA ligase catalyzes the reaction and reconnects the cleaved plasmids with the DNA strands by creating covalent bonds

Stage 4: Isolation of transformed cells

-The recombinant DNA is then inserted into the host cell

-The cells that are transformed to contain the recombinant DNA are separated from untransformed cells

Crime Scene Investigation

1) Explain the content of the human genome: % protein encoding, % regulatory, % STRs AND distinguish locus from gene from alleles

-There is less than 2% protein encoding (code for proteins) in the human genome, around 3% STRs (short sequences of non-coding DNA), and around 8% regulatory genes that control gene expression

http://upload.wikimedia.org/wikipedia/commons/thumb/6/64/Components_of_the_Human_Genome.jpg/370px-Components_of_the_Human_Genome.jpg

 -A locus is the position of a gene on a chromosome

-A gene is the specific part of DNA base pairs on a chromosome that code for a phenotype

-An allele is a different type of gene in the same locus

http://www.csulb.edu/~kmacd/361-6-Ch1_files/allele.jpg

2) Explain the use of STRs in profiling to demonstrate the rarity of sharing all/most/many STRs

STRs (short tandem repeats) are used in DNA profiling because they can be highly polymorphic, which means there are many varieties of it. Therefore, STRs are very unique to each individual and therefore it can be used to discriminate between individuals esp. at a crime scene. 

3) State and describe the evidence of the gel to identify the suspect at the crime scene by referring the alleles found at the crime scene and the allele of the suspect(s)

-On the gel, the second column is the DNA found in the crime scene, which is subsequently followed by Suspect A in column three, Suspect B, Suspect C and Suspect D in the next three columns.

-As you can see below, the DNA found at the crime scene is exactly in line with Suspect C of the fourth column. This is because the alleles (one blue dash is an allele) exactly correspond

DNA Profiling

Write a brief description of the difference between RFLP and STRs

-RFLP (restriction fragment length polymorphism)- it indicates a difference between samples of homologous DNA molecules via restriction enzymes

-STRs (short tandem repeats)- they consist of short repeated sequences of DNA (2-8 nucleotides) and are located on non-coding regions of chromosomes. STRs are also polymorphic, which means that there are nine or more different types of alleles that can be detected.

-The STR and RFLP methods differ in DNA profiling and the STR has more advantages, which is why it is preferred today. While the STR only needs a very small amount of DNA to run, the RFLP does not as it requires a very large sample of DNA to work. Therefore, with a small amount of DNA, using the STR method reduces the likelihood of destroying sample due to the utilization of PCR. On the other hand, the RFLP method destroys the cell after it is completed. Furthermore, the STR method takes only around a couple hours to complete whereas the RFLP method could take weeks. STR is also more accurate than RFLP because STR targets specific sites unlike RFLP.

How many STRs does the FBI use to analyze?

-They use 13 different STRs, with repeats of four or five nucleotides, plus sex markers

Describe how PCR is used in DNA profiling

-PCR is used to amplify the specific regions of the DNA (STRs) and is therefore used to ultimately detect differences between STRs. During PCR, DNA fragments, with primers, are heated and cooled to activate primers and DNA polymerase in copying the fragments over and over again until the amount of copied fragments are "amplified"

Describe how electrophoresis is used in DNA profiling

-Electrophoresis is used to separate different DNA fragments (which were amplified with PCR) by putting the fragments onto polyacrylamide gel. The polyacrylamide gel in PCR separates different DNA fragments by size and the smaller the fragment, the further it travels through the gel.

Explain how the probabilities of having a specific set of STRs is extremely unlikely. Therefore if yours is present at a crime scene, what does that mean?

-The probability of having a specific set of STRs is extremely unlikely because one in every 10^-100 quadrillion share the same STR profile. The number is greater than the number of humans that ever lived, therefore each STR profile is specific to one person. If my STR is present at a crime scene, this means that you were definitely at the crime scene but does not guarantee any more than that.

Recombinant DNA Techniques

Extracting DNA

Reasons to Collect DNA

-Genetic testing

-Body identification

-Analysis of forensic information

Outline and explain the purpose of: swabbing, lysis buffer, centrifugation, and isopropyl alcohol

-Swabbing is the process of collecting a sample of cells. An example of this is swabbing the inside of somebody’s mouth to collect cells for DNA extraction.

-The lysis buffer contains detergent and enzyme proteinase K and separates the DNA from the cell. The detergent breaks down the cell membrane and nuclear membrane so that the cells would burst open and release the DNA. The enzyme cuts down the histone proteins, which the DNA is wrapped around, and therefore frees the DNA.

-Centrifugation causes the remaining debris and protein to sink to the bottom of the test tube while the DNA remains distributed in the liquid. This thus separates the DNA from any debris and protein, which may interfere with analysis.

-Centrifugation can also be used after isopropyl alcohol is mixed with the DNA so that the DNA would sink to the bottom of the tube, allowing the DNA to be isolated.

-Isopropyl alcohol concentrates the DNA together so that it is visible to the naked eye. This is because DNA is not soluble in isopropyl alcohol.

Cutting DNA

Explain how enzymes cut DNA

-Enzymes that cut DNA are called restriction enzymes and they are mostly found in bacteria. These enzymes are sequence specific, which means that they only bind and cut specific DNA sequences. After the restriction enzymes bind to a specifically recognized sequence, they cut down the sugar phosphate backbones of the DNA strands.

Find and compare 3 different enzymes (one which must cut with ‘sticky ends’ and another with ‘blunt ends’)

-AluI (source: Arthrobacter luteus) produces blunt ends

-BamHI (source: Bacillus amyloliquefaciens) produces sticky ends

-EcoRI (source: Escherichia coli) produces sticky ends

-AluI, which produces blunt ends, cuts the strand symmetrically whereas BamHI and EcoRI, producing sticky ends, cut the strand asymmetrically. This means that BamHI and EcoRI create 5’ or 3’ overhangs on the DNA strand, which makes it easy to ligate. On the other hand, enzymes such as AluI leave no overhang because it cuts the strand symmetrically.

Separating nucleic acids/protein

Outline and explain steps: purpose of gel, electricity, ‘running buffer’, DNA standard, ‘loading buffer’, ethydium bromide

-The purpose of gel is to sort out DNA strand by filtering them with its sponge-like consistency.

-The electricity during electrophoresis makes the DNA strands in the wells of the gel move through the gel via a current. The smaller the strand, the further it will go in the gel.

-Running buffer is mixed with agarose when making the gel so that electrical charges can flow through the gel

-DNA standard contains DNA strands with known lengths; therefore it is used as a reference to compare the strands from the DNA sample with those from the standard.

-A loading buffer contains a dye that makes the DNA sample visible as it goes through the gel. Furthermore it makes the DNA sample thicker so that it will stay in the gel well instead of floating away.

-Ethydium bromide is used to stain the DNA sample because it binds to the DNA and is visible under fluorescent light.

Amplifying DNA

Outline and explain steps: role of primers, heating, cooling, reheating, over and over, compare amount of DNA at start to amount of DNA at finish.

-The primers in PCR attach to sites on the DNA strand that are at either end of the segment that is targeted to be copied.

-Heating the DNA causes it to unwind into two separate strands. Cooling it then leads to the primers in the solution to attach to the targets before the two DNA strands can rejoin. The solution gets heated again, causing the DNA polymerase to attach on a primer and add complementary nucleotides onto the strand. The temperature is then heated, cooled, and heated again to repeat this cycle, creating more complementary strands of the target sequence. After 4 cycles, there are 8 fragments that contain only the target sequence. After 30 cycles, there are over a billion fragments that contain only the target sequence and 60 copies of the longer length molecules.