DNA Replication worksheet. 1. Define the following : a. DNA b. DNA Replication c. Complimentary strand d. Primer e. DNA polymerase f. Okazaki Fragment 2. Why do we say DNA replication is semi-conservative? 3. If a single copy of double stranded DNA molecule was to undergo replication, how many copies of double stranded DNA will be present after a. 2 rounds of replication 4 copies of double stranded DNA b. 3 rounds of replication 8 copies of double stranded DNA c. 4 rounds of replication 16 copies of double stranded DNA 4. PCR (Polymerase chain reaction) is a technique routinely used in research laboratories to amplify specific regions within DNA. The idea behind the technique is to enable artificial DNA replication targeted to regions of interest within the DNA. a. Listen to your TA explain how PCR works and make notes. b. Based on your notes, answer the following question. (i) Which of the following PCR reactions will work and why? Reaction A Reaction B Reaction C Reaction D DNA YES NO YES YES DNA polymerase NO YES YES YES Primers YES YES YES YES dNTPs YES YES YES NO PCR reaction Buffer YES YES YES YES Key: Reaction C because only C has all the ingredients needed for PCR to work (ii) if we start with 25 copies of double stranded DNA molecule, after three rounds of PCR how many molecules of DNA will be present? Key : After 1 round we will have 25 X 2 = 50 double stranded DNA molecules After 2 rounds we will have 50 X 2 = 100 double stranded DNA molecules After 3 rounds we will have 100 X 2 = 200 double stranded DNA molecules Another method using the formula : 2n= no of strands after n rounds of replication of a single double stranded DNA molecule X (2n) = no of strands after n rounds of replication of X double stranded DNA molecules Here X= 25 ; n= 3 25 (23) = 25 (8) 200 double stranded DNA molecules (iii) If the DNA polymerase used in the PCR reaction adds 900 nucleotides in one minute how many seconds will it take for the enzyme to complete replication of DNA that is 4.6 kilo bases long (4600 nucleotides long)? Key : Length of DNA that needs to be replicated = 4600 nucleotides Speed at which the DNA polymerase adds nucleotides = 900 in 1 minute = 900 in 60 seconds = 900/60 nucleotides in 1 sec = 15 nucleotides in 1 sec Time required to replicate 4600 nucleotides = 4600/15 seconds = 306.67 seconds. 5. In humans, only one-tenth of a single percent of total DNA differs from one person to the other. Scientists can use these variable regions to generate a DNA profile of an individual, using samples from blood, bone, hair, and other body tissues and products. In criminal cases, this generally involves obtaining samples from crimescene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions that are unique for each individual (markers). Often when the sample obtained is very small or damaged PCR is used to amplify the signal to enable detection. a. The minimum number of copies of double stranded DNA that can be detected by PCR based method is 128. If double stranded DNA was extracted from a sample at a crime scene it required 7 cycles of PCR to be detected, how many copies of double stranded DNA was retrieved from the original sample? Key : Minimum detectable amount of double stranded DNA = 128 molecules No of cycles need to reach that number = 7 Let the number of double stranded DNA retrieved be = X molecules We know that the number of strands of DNA after ‘n’ round of replication of a single double stranded DNA is given by 2n = no of double stranded molecules formed after n rounds of replication. Where ‘n’ is the no of cycles. Here, there is an unknown “x” number of initial double stranded DNA present. = X (2n)= 128 in this case we know that n = 7 (given) => X (27)= 128 we know that, (27) = 128 X = 1. Answer: 1 double stranded DNA was retrieved from the original sample. 6. During normal DNA replication in the cell, it is important to eliminate inappropriate insertions of nucleotides (errors), as they will result in a change in genetic material of that organism (mutation). It has been found that a eukaryotic DNA polymerase has an error rate of 1 X 10-8 nucleotides. a. If this polymerase were used to replicate the entire human genome comprising 3.3 X109 bases, how many mutations would be introduced in the human genome? Key : According to the question, the polymerase on an average introduced 1 error in every 108 nucleotides Going by that rate if it replicates 3.3 X 109 bases it will introduce = 3.3 X 109 /108 = 3.3 X 10 = 33 errors or mutations b. How does an organism deal with and prevent introduction of mutation during replication?