You use the primer 5^' GCCTCGAATCGGGTACC 3^' to sequence...

You use the primer $5^{\prime}$ GCCTCGAATCGGGTACC $3^{\prime}$ to sequence part of the human DNA insert of a recombinant DNA molecule made with a plasmid vector. The result of the automated DNA sequence analysis is shown here. The height of the peaks is unimportant. a. Write the sequence of all the nucleotides of human DNA that you can determine. Indicate the $5^{\prime}-$ to- $3^{\prime}$ orientation of this sequence. b. Is the sequence you wrote in (a) part of the new DNA strand that was synthesized in the sequencing reaction or part of the template strand used in the sequencing reaction? c. How did you know how to design the primer you would need for the sequencing reaction? Diagram the recombinant DNA molecule to be sequenced, indicating the human and vector sequences, the position and orientation of the primer, and the position and orientation of the new DNA that would be synthesized during the sequencing reaction, using Fig. 9.7 as a guide. d. Show the full sequence of the smallest DNA molecule that would be synthesized in the sequencing reaction and that would contain dideoxyG (ddG). Indicate the $5^{\prime}-$ to-3 $^{\prime}$ orientation of this molecule and the location of the ddG. e. How would the data differ from that shown if you accidentally left the dATP out of the reaction?

You use the primer $5^{\prime}$ GCCTCGAATCGGGTACC $3^{\prime}$ to sequence part of the human DNA insert of a recombinant DNA molecule made with a plasmid vector. The result of the automated DNA sequence analysis is shown here. The height of the peaks is unimportant.
a. Write the sequence of all the nucleotides of human DNA that you can determine. Indicate the $5^{\prime}-$ to- $3^{\prime}$ orientation of this sequence.
b. Is the sequence you wrote in (a) part of the new DNA strand that was synthesized in the sequencing reaction or part of the template strand used in the sequencing reaction?
c. How did you know how to design the primer you would need for the sequencing reaction? Diagram the recombinant DNA molecule to be sequenced, indicating the human and vector sequences, the position and orientation of the primer, and the position and orientation of the new DNA that would be synthesized during the sequencing reaction, using Fig. 9.7 as a guide.
d. Show the full sequence of the smallest DNA molecule that would be synthesized in the sequencing reaction and that would contain dideoxyG (ddG). Indicate the $5^{\prime}-$ to-3 $^{\prime}$ orientation of this molecule
and the location of the ddG.
e. How would the data differ from that shown if you accidentally left the dATP out of the reaction?

Key Concepts

Recombinant DNA Molecules

Recombinant DNA molecules are engineered by combining DNA from different sources, typically involving a vector (like a plasmid) and an insert (such as a gene or DNA fragment from another organism). Understanding the arrangement and orientation of vector and insert DNA is crucial for experiments involving sequencing or cloning.

DNA Sequencing

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It forms the basis for understanding genetic information, and techniques in sequencing allow researchers to analyze genes, map genomes, and diagnose mutations.

Sanger Sequencing (Chain Termination Method)

Sanger sequencing is a method that uses dideoxynucleotide triphosphates (ddNTPs) alongside normal deoxynucleotide triphosphates (dNTPs) to terminate DNA synthesis at specific bases. Because ddNTPs lack a 3' hydroxyl group, their incorporation produces fragments of various lengths that, when separated, reveal the DNA sequence.

Primer Design

Primer design involves selecting a short, single-stranded oligonucleotide sequence that is complementary to a known region adjacent to the target sequence on the template DNA. This primer provides a starting point for DNA polymerase to begin synthesis, ensuring that the reaction is specific to the region of interest.

Template versus Newly Synthesized Strand

In sequencing reactions, it is important to distinguish between the template strand, which serves as the pattern for synthesis, and the newly synthesized strand, which is produced by DNA polymerase. Sequencing data is derived from the newly synthesized strand while its sequence reflects the complementary information to the template.

Chain Termination Using Dideoxynucleotides

Dideoxynucleotides (ddNTPs) are modified nucleotides that cause termination of DNA synthesis because they lack the 3' hydroxyl group needed for chain elongation. Their incorporation at random positions during DNA synthesis generates fragments of different lengths that can be resolved to deduce the original sequence.

Sequence Orientation (5? to 3? Directionality)

DNA sequences are always read and synthesized in the 5? to 3? direction. This orientation is critical in interpreting sequencing results, designing primers, and understanding the action of DNA polymerase during replication and sequencing reactions.

Effect of dNTP Omission in Sequencing Reactions

The omission of a specific dNTP (for example, dATP) in a sequencing reaction will prevent the incorporation of that nucleotide during chain elongation. This leads to gaps or altered fragment lengths in the generated data, which can result in incomplete or skewed sequence information.

Transcript

00:02 So, one of the following sequences are obtained from a clone piece of genome that includes part of two axons of a gene.

00:09 The other sequence was obtained from the corresponding part of a cdna clone, representing the mrna for this gene, which means the mature mrna after processing or splicing.

00:20 So obviously, for simplicity, the intro is unrealistically short, but it also has other features needed for the splicing to happen.

00:31 So for this sequence, i have copied them on a board already.

00:35 From one of it is longer, the longer sequence from 5 to 3 from left to right.

00:41 And the other sequence, number 2, is shorter, so i copy it reversely from 5 to 3 the other side.

00:49 There's a reason for me to do this later.

00:51 So the first question is that which sequence is a genomic fragment and which is a cdna fragment? so to answer the first question, obviously, a, the longer one, number 1, is the genomic.

01:11 It's genomic dna because it gave rise to a pre -mrna, so the pre -mrna still has the intron.

01:29 So that's why it's longer.

01:30 Now number 2, shorter one, is cdna because cdna comes from the mature mrna and it has axons only.

01:53 The introns are being cut away during splicing, so that's why it has less nucleotide sequence.

01:59 So that answers the first question.

02:01 B, write the rna -like strand of the genomic dna, indicator 5 and 3, and also draw a vertical line between the bases that are axon and intron boundary.

02:14 So for the second question, b, i'm going to actually move it up here underneath the genomic dna to make it easier for you.

02:23 So now i'm going to use red color to write down the mrna or pre -mrna according to genomic dna.

02:31 So in that case, we have the 5 ' end on the right -hand side.

02:38 It follows, it's mrna, i'm sorry, there's no t.

02:43 So it follows base pair rule, a pair up with u and g pair up with c.

02:54 All right, and then mrna is complementary to the genomic dna when it's being made, which means they have opposite orientation.

03:02 And that's why the 5 ' end is on the right side.

03:05 So u, a, a, c, u, g, a, u, u, c, u, a, g, g, c, u, a, c, u, c, u, u, u, c, a, c, c, u, a, 3.

03:50 So remember, this is the pre -mrna before b, it is being spliced.

03:57 So before splicing happens, so this is longer than the cdna, which is this one here.

04:02 The cdna comes from the mature mrna that is being spliced.

04:07 So it doesn't have intron, but the red sequence here still has intron.

04:11 So to actually define the region of intron and axon boundary, we must line up the two sequences to see which sequence are missing actually in the pre -mrna.

04:29 So after i line up the two sequences, so i'm lining up this sequence here and this sequence here and then try to see if they match and if any of the sequence are actually missing.

04:44 And again, since it's an mrna and down here is a cdna, it's a dna form.

04:51 So whenever you have a t in the dna, that actually match up with a u in mrna.

04:59 Okay, so as you can see, the first several sequence are the same until you hit this c here.

05:11 So you can see that they actually match all the way to here.

05:17 And then the c, u, a, gg, cc, ua, they are actually missing.

05:29 So then the next sequence is going to be right here.

05:34 So as you can see that the next one after that is uc, u, uu, u, ca, cc, ua is going to match the other half of the cdna.

05:45 But this sequence that in the middle is actually missing.

05:49 So we know that the first part of the sequence, this is the exon one and this part actually match this part is actually exon two.

06:08 And then in between is intron...

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