Lab 5: DNA Isolation and Amplification

The purpose of Chelex in this lab is to (select all that apply):

-Prevent the DNA from being degraded -Bind to the Mg2+ in the DNA extract

During the wet lab portion of the DNA Isolation lab you must wear

-Safety goggles -Lab coat -Standard lab attire -Gloves

Which one of the following statements on BLAST search is NOT true?

-The E value indicates the similarity between your sequence and the sequences in the database, the higher the E value the better the match -The higher the E-value, the more likely your sequence and the sequence in the database are a true match

All of the following characterize a good PCR primer except a. similar melting points for forward and reverse primers b. 100-110 base pair length c. lack of consecutive identical base pairs d. high Guanine and Cytosine content e. 20-30 base pair length

100-110 base pair length

When designing a primer, the primer length is ideally between _____ and _____ base pairs long.

20 and 30

While using the BLAST search program, you input a eukaryotic nucleotide sequence into blastx (nucleotide to protein sequence. which results in no "hits." Which of the following could be reasons that would explain this? I. The nucleotide sequence was part of a promoter region II. The nucleotide sequence encoded for an intron III. The nucleotide sequence encoded for an exon.

I and II

What is the special property of Taq polymerase that allows it to work in PCR?

It is heat resistant

What is the importance of using Taq Polymeraase when conducting PCR?

It is very heat resistant and is thus resilient to denaturation.

What is the importance of constructing a phylogenetic tree of human mitochondrial DNA?

It shows the evolutionary history of female mitochondrial DNA from its origin in Africa

In BLAST which of the following signify the best sequence homology?

Low E-Value, High Score

Which is the correct order of the PCR reaction?

Melting of DNA strands, annealing of primers, extension of new strand

Isolated DNA is susceptible to enzymes that degrade DNAses. They require the cofactor ________ which the chelex solutionbinds to tightly.

Mg2+

During PCR, the sequencing primer is needed for:

The sequencing primer is not needed for the PCR process to proceed.

In the PCR lab, what is the purpose of the Chelex solution?

To prevent DNA degradation.

In the DNA Isolation lab, you must wear gloves in order to prevent any of your cells from contaminating other students' samples.

True

mtDNA is more abundant per cell than nuclear DNA.

True

All of the following are true regarding DNA isolation and amplification except: a. Chelax prevents DNA degradation by binding all of the Mg2+. b. We looked at a protein-coding region that can collect many mutations without losing function. c. Mitochondrial DNA is inherited just from the mother because the mitochondria in the sperm is destroyed after fertilization. d. The sequencing primer in PCR is specific to a region that needs to be amplified. e. Taq Polymerase is used in PCR because it is heat resistant and can resist degradation.

We looked at a protein-coding region that can collect many mutations without losing function.

Inherited nuclear and mitochondrial DNA are respectively carried by:

X and Y chromosome; maternal mitochondria

If your mother has haplotype B and your father has haplotype J what haplotype are you?

You are haplotype B because mitochondrial DNA is inherited from your mother only.

Which of the following is false regarding polymerase chain reaction? a. a mismatch on the 3' end of the primer prevents RNA polymerase from extending DNA b. the forward strand is the template strand for the forward primer c. a good primer anneals to a hypervariable region d. the reverse primer anneals to the antisense strand e. human RNA polymerase would be denatured in each cycle of PCR

a good primer anneals to a hypervariable region

In mitochondrial DNA isolation lab, if the DNA is susceptible to DNases, what is the best solution to prevent this problem?

add chelex; because it binds to Mg2+ in the extract

What is the purpose of PCR?

amplifying DNA

In what order do the three main steps in each cycle of PCR occur?

denaturation, annealing, extension

What elements within a haplogroup are found to be similar? a. RNA b. DNA c. haplotypes d. phenotypes e. genotypes

haplotypes

The function of the Chelex beads in PCR is to

prevent DNA degradation by binding to Mg2+

Primers in PCR will always be:

single-stranded DNA

When working with the BLAST program results, what does the E (Expect) Value of a hit represent?

the number of hits one can expect to see by chance when comparing a sample to a database of a particular size

Forensic laboratories occasionally use mitochondrial DNA comparison in order to establish identification of individuals, human remains, and more notably, older unidentified skeletal remains. Which of the following reasons best serves as an advantage when using mtDNA as opposed to nuclear DNA during such analyses?

A greater number of copies of mtDNA per cell increase the chance of obtaining a useful sample.

Which of the following statements concerning restriction digests and BLAST in Lab 5 is FALSE? a. A low score and a high E-value are desirable when comparing your DNA sequence to a translated protein b. Ethidium bromide allows visualization of bands c. Ambiguities in your DNA sequence are represented by any symbol that is not a capital T,G,C, or A d. Under complete digestion, a plasmid with an insert will yield 2 bands e. The sequence CCCTT should always be removed before using BLAST as it is a cloning vector

A low score and a high E-value are desirable when comparing your DNA sequence to a translated protein

Which of the following statements is true? a. A haplogroup comprises haplotypes from separate ancestral lineages. b. DNA sequencing involves the use of primers and Taq polymerase. c. Dideoxynucleotides have a hydroxyl in the 3' prime position instead of a hydrogen. d. Mitochondrial DNA types (sequences) are termed haplotypes because of the fact that they are inherited solely from the father. e. A single nucleotide change in the DNA sequence of an organism is referred to as an SNP.

A single nucleotide change in the DNA sequence of an organism is referred to as an SNP.

Which of the following is true about the role of the sequencing primer? a. It indicates the area of DNA to be amplified when doing a PCR. b. It is used before PCR to ensures that the sequence of forward and reverse primers are adequate. c. After PCR has been completed, the sequencing primer is used to determine the order of DNA nucleotides in a given segment of DNA. d. It guides the forward and reverse primers to specific sequences on the DNA. e. It ensures that no other location in the genomic code has the same sequence.

After PCR has been completed, the sequencing primer is used to determine the order of DNA nucleotides in a given segment of DNA.

PCR:

Amplifies specific DNA sequences.

Mitochondrial DNA is used to follow maternal inheritance and sequences on the Y chromosome is used to follow paternal inheritance. Why is it not practical to analyze paternal inheritance in this class?

Because only men have a Y chromosome, the women in the class wouldn't be able to participate

Why is Mitochondrial DNA only used to trace maternal lineage?

Because the sperm did not contribute its mitochondria upon fertilization.

In the DNA Isolation and Amplification lab, when blasting, which of the following best tells us that the sequence is a good match?

Blast shows that the match has a high Score and a low E-value.

In this lab, DNA will be collected from

Cheek cells/saliva

According to the LS Core Lab Safety Sheet for this lab, which of the following is a potential hazard you will face in the DNAIsolation lab?

Chemicals causing eye irritation

Which of the below statements IS true about the process of PCR? a. During PCR, the primers will be extended from the 3' end. b. The reverse primer is extended in the 5' to 3' direction following the reference strand. c. The reverse primer is identical to the reference strand. d. The forward primer binds to the reference strand. e. During PCR, the primers will be extended from the 5' end.

During PCR, the primers will be extended from the 3' end.

Mitochondrial DNA can be inherited from either an individual's father OR mother.

False

You can remove your lab coat once you are done with the wet lab portion of the DNA Isolation experiment, even if other people in the room are still working with Chelex.

False

When considering DNA isolation and amplification, one must design primers in order to run them through PCR for amplification. Which of the following would be best the follow when deciding whether or not your primer of interest should be used? a. Adenine and Cytosine should make up 10 - 20% of the total base pairs. b. Guanine and Thymine should make up 30 - 40% of the total base pairs. c. Guanine and Cytosine should make up 50 - 60% of the total base pairs. d. Adenine and Thymine should make up 50 - 60% of the total base pairs. e. Guanine and Cytosine should make up 30 - 40% of the total base pairs.

Guanine and Cytosine should make up 50 - 60% of the total base pairs.

When designing primers for use in PCR, which of the following base pair compositions is considered the best?

Guanine and Cytosine should make up 50 - 60% of the total base pairs.

What are the qualities of a good primer? a. Ending the primer in a A or T b. High G-C content and no longer than 20-30 bases c. Extremely high Tm d. Long Strings of Repeating Nucleotides e. Palindromic sequences

High G-C content and no longer than 20-30 bases

in the DNA isolation and Amplification lab, if a student wants to conduct PCR for a region of DNA known to have 90% GC content at the site of Primer hybridization, what experimental settings should she set and what materials must she add in order to have optimal results?

High annealing temperature; Reverse and Forward Primers, Thermophillus Aquaticus (Taq) DNA Polymerase and dNTPs

Mitochondrial DNA (mtDNA) is frequently amplified for analysis when the samples are old (in museum or archeological finds) or in small amounts (in forensics). What is/are the advantages of using mtDNA over nuclear DNA? I. mtDNA is more stable II. mtDNA is more abundant per cell than nuclear DNA III. mtDNA is less abundant per cell than nuclear DNA

I, II, and III

In the DNA isolation and amplification lab, the Primer3 program was used to generate primer pairs. Choose the best answer from the following about primer design: a. If one primer has a much higher melting temperature than the other, one strand of the target mtDNA sequence will be amplified more than the other. b. A good primer should have the same sequence as the template strand of the mtDNA. c. A good primer should be composed of an equal number of all 4 deoxynucleotide triphosphates. d. The primers in a primer pair should have similar sequences so that they can anneal to each other. e. A good primer should anneal to a region of the mtDNA sequence with a high level of variability in order to identify the individual's specific haplotype.

If one primer has a much higher melting temperature than the other, one strand of the target mtDNA sequence will be amplified more than the other.

Which of the following is false? a. Mitochondrial DNA sequence can be used to probe our maternal lineage. b. A good primer will hybridize to highly conserved areas of this region. c. The D-loop region is a good choice because it is a control region and can thus accumulate more mutations without affecting function. d. In PCR, RNA polymerase is used to make copies of the region between the primers. e. In PCR amplification, changes in temperature are used for DNA denaturation and hybridization to primers.*

In PCR, RNA polymerase is used to make copies of the region between the primers.

Following statements describe PCR (Polymerize Chain Reaction). Choose one that is true. a. Different from DNA sequencing, PCR is based on in vitro DNA synthesis while sequencing is not. b. This protocol used 5 separate DNA polymerization reactions taking place in 4 reaction tubes. c. For PCR, dideoxynucleotides have a hydrogen in the 5' position. d. The fragments in different reaction tubes would be separated on a denaturing polyacrylamide gel and detected by either autoradiography or Ethidium Bromide. e. In the case of PCR, the synthesis of new DNA strands is not the same as DNA sequencing reaction where the new strands are terminated prematurely

In the case of PCR, the synthesis of new DNA strands is not the same as DNA sequencing reaction where the new strands are terminated prematurely

Which of the following is not true about PCR? a. It is used to amplify RNA b. It is used to amplify a small amount of DNA c. Taq polymerase is used d. 2 primers are needed e. It is an in vitro synthesis

It is used to amplify RNA

Primer mismatches affect PCR. When there are mismatches at the 5' end or the center, the primers can still prime the PCR by changing the ____________. However, when there are mismatches at the 3' end, _____________ and _______________.

Melting temperature; the RNA polymerase cannot extend; polymorphic sites will not bind well.

Which statement is in support of the Endosymbiotic theory?

Mitochondria have their own DNA

Which of the following is true about mitochondrial DNA? a. Mitochondrial DNA is a double helix like nuclear DNA. b. Only the mother contributes to the mitochondrial genome because sperm are destroyed after fertilization. c. Mitochondrial DNA encodes proteins, tRNAs, and mRNAs d. There are less copies of mitochondrial DNA than copies of nuclear DNA in a cell. e. Mitochondrial DNA is present in all living organisms.

Mitochondrial DNA is a double helix like nuclear DNA.

Does the mitochondrial DNA sequence amplified in the experiment code for functional proteins?

No. The sequence is located in the control region, which does not code for functional proteins.

What are differences between mitochondrial and nuclear DNA?

One is linear, one is circular.

Which of the following is false regarding Polymerase Chain Reaction (PCR)? a. PCR requires a heat-resistant DNA polymerase like Taq polymerase. b. Approximately one billion copies of the target sequence are synthesized. c. Three primers hybridize to the DNA sequence. d. PCR is performed in a test tube, or in vivo.

PCR is performed in a test tube, or in vivo.

Which of the following is true about PCR? a. PCR is simply an in vivo DNA synthesis reaction repeated many times. b. PCR stands for polypeptide chain reaction. c. PCR is used to amplify the region we want to sequence. d. PCR used double stranded DNAs, usually 20-35 bases long, called primers.

PCR is used to amplify the region we want to sequence

Which of these descriptions of PCR is not correct?

PCR requires a single primer

What is the technique used for DNA amplification?

Polymerase Chain Reaction

Which of the following is most affected by a mismatch in the 3' end of a primer with a DNA strand?

Primer Efficiency

Which of the following is not a characteristic of a well made primer? a. Guanine and Cytosine make up 50-60% of the primer b. Primer is between 20-30 base pairs long c. Primer ends at 3' in a G or C d. Primer anneals to Numt DNA e. Not having many of the same base pairs in a row

Primer anneals to Numt DNA

Which of the following statements regarding primers is true? a. Primers are single-stranded RNAs, usually 20-30 bases long. b. Good primers should have long sequences (more than 40 base pairs). This will make it easier for a primer to bind to DNA more tightly. c. Adenine and Thyamine should make up the majority of the total base pairs in a good primer. d. Primer specificity is increased by making the 3' (C terminmus) end in a G or C, since G and C bind more tightly than A or T. e. Good primers are designed to bind to hyper variable regions of the DNA that we want to amplify.

Primer specificity is increased by making the 3' (C terminmus) end in a G or C, since G and C bind more tightly than A or T.

Which one of the following statements is FALSE for primer design in order to amplify a very specific target DNA region? a. Primers should between 4-30 base pairs long. b. Guanine and cytosine should make up 50-60% of the total base pairs. c. Primers should end (3') in a G or C, or CG or GC. d. Primers should not contain long repeating basepairs (six or more of the same base in arow). e. Melting temperatures should be between 55-80 degrees C.

Primers should between 4-30 base pairs long.

Which one of the following statement should not be in the set of rules for primer design? a. Primers should between 20-30 base pairs long. b. Melting temperatures should be between 55-80 C. c. Guanine and cytosine should make up 50-60% of the total base pairs. d. Primers should end (3') in a G or C, or CG or GC. e. Primers should contain long runs (four or more of the same base in a row).

Primers should contain long runs (four or more of the same base in a row).

Which one of the rules is not correct for the primer design? a. Guanine and cytocine (G and C) should make up 50-60% of total base. b. Primers should end (5') in a G or C, or CG or GC c. The melting temparature should be between 55-80 C. d. Four or more of the same base in a row can result in mispriming. e. Primers should be between 20-30 base pair long.

Primers should end (5') in a G or C, or CG or GC (they should end 3')

When picking a good pair of primers, there are several rules that need to be considered. Which of the follow is a correct rule for designing good primers? a. Primers should end in a G or C, CG, or GC because this increases primer specificity. b. Primers should be between 10-17 base pairs long so that melting temperatures can be lower. c. Primers should end in a polymorphic region because they will bind better. d. Guanine and cytosine should ideally make up approximately 60-70% of the total base pairs because they allow for stronger binding. e. Melting temperatures for the two primers should be as dissimilar as possible since they are annealing to different strands.

Primers should end in a G or C, CG, or GC because this increases primer specificity.

Which of the following components is NOT necessary for a typical PCR reaction to initiate/proceed? a. Protein kinase b. Forward primer c. Reverse primer d. Taq polymerase e. DNA template

Protein kinase

Once you have loaded your sample on the plate you can dispose of the tube containing your cheek cells by:

Putting it in the regular trash.

Assume that instead of Taq polymerase, another polymerase, one that is very heat-sensitive, was used in a PCR. What differences would one observe when this new experiment is compared to the original experiment (one that includes Taq polymerase).

There would be little to no PCR product because the new polymerase would denature and lose its function in the reaction..

Which of the following is not required for PCR? a. Taq Polymerase b. Buffer solution c. Deoxynucleoside triphosphates d. Two primers that bind to denatured genomic DNA at complementary sequences e. A sequencing primer

Sequencing primer

Which of the following is TRUE regarding DNA sequencing and amplification: a. PCR is an in vivo DNA synthesis reaction repeated many times. b. Taq polymerase is resistant to denaturation caused by heat. c. Mitochondrial DNA is amplified using the Chelex technique. d. Chelex prevents DNA degradation by binding Ca 2+. e. Mitochondrial DNA can trace paternal lineage.

Taq polymerase is resistant to denaturation caused by heat.

When looking at a hit list from a Blast on the NCBI website what does the E value indicate? if you were to incrase the sequence length Blasted how would that effect its E value?

The chance that the hit would randomly match the sequence. Decrease.

What is the purpose of adding dNTPs to the PCR set up?

They act as the building blocks from which DNA polymerase can synthesize new strands.

In this lab, we used polymerase chain reaction (PCR) in order to amplify our DNA sequence. In order to have proper amplification, we needed to produce a good primer to hybridize on both ends of our desired DNA sequence for amplification. Which of the follow was NOT a parameter that would make for a "good" primer? a. Avoid runs of four or more of the same base in a row because of mispriming. b. Primers should be approximately 20-30 base pairs long. c. Guanine and cytosine should make up at lesat half of the total base pairs. d. The melting temperature of the primers needed to be at most 50 degrees celcius or higher so that it anneals and dissociates at proper temperatures.

The melting temperature of the primers needed to be at most 50 degrees celcius or higher so that it anneals and dissociates at proper temperatures.

Which of the following can be determined by studying the control region of your mitochondrial DNA? a. The presence or absence of nonsense mutations that will alter proteins critical to cellular respiration b. The presence or absence of base pair deletions that will alter proteins critical to cellular respiration c. The presence of mutations specific to your maternal ancestor's haplotype group d. Your father's country of birth e. The presence or mutations specific to all your ancestors' haplotype groups.

The presence of mutations specific to your maternal ancestor's haplotype group

Other than ddNTPs, DNA Polymerase, and purified plasmid DNA, what should be added to reaction tubes in the sanger method of sequencing? What type of gel should be used to load the samples?

The primer with a 5' fluorescent tag attached to it needs to be added. Polyacrylamide gel to be used to load the samples.

What would most likely happen if only one oligonucleotide primer was used in PCR?

The primer would amplify only one strand so that double stranded DNA would not be produced.

During PCR, the sequencing primer is needed for: a. To help denature the DNA strand so the primers can bind. b. As a back-up for the forward primer. c. The sequencing primer is not needed for the PCR process to proceed. d. As a back-up for the reverse primer.

The sequencing primer is not needed for the PCR process to proceed.

In the DNA Isolation and Amplification lab, we must amplify a region of mitochondrial DNA by using a technique called Polymerase Chain Reaction (PCR) ________.

because we need many copies to sequence the DNA