DNA Sequencing

Question 1

What is Sanger sequencing

Answer 1

It is a way to read the sequencing of genome

Question 2

How is Sanger sequencing carried out

Answer 2

1. Create number of copies of the genome using PCR
2. Using a primer, we add some initial nucleotides which then can allow the DNA to add nts afterwards
3. The trick here is that some of the nts that DNA pol adds cannot be extended upon
4. As we extend, we get fewer and fewer templates, giving us information for the each base that was added on

This process is done to read a strand of DNA template, usually couple thousand base pairs long

Question 3

What is microarray

Answer 3

It is a modern, second generation method to investigate perhaps the amount of DNA present in a sample

Question 4

Describe the process of microarray

Answer 4

DNA from the person of interest (sample) and DNA from control is taken, it is chopped up or its RNA can be chopped up, they are added to a plate. On this plate afterwards, a laser is shown which can then tell us how much control DNA and sample DNA is present in comparison. So if the person has Down’s syndrome, the sample DNA would be more in quantity than the control DNA

Question 5

What are the restriction of using the method of microarray

Answer 5

1. This method doesn’t allow us to copy the entire genome
2. Cant check SNP
3. Might miss small additions or deletions

Question 6

What are the advantages

Answer 6

It is an easy and convenient way to measure if there has been an addition or deletion at some part of the genome

Question 7

What is 2nd generation sequencing

Answer 7

1. Sample DNA is taken and chopped up.
2. At the end of these pieces of DNA we add adapters to stabilize the individual pieces of DNA. These fragments are 200 bps to 1000 bps long
3. Amplify the amount of DNA by PCR
4. It is placed on a channel which have control pieces of DNA sticking out
5. The sample DNA pieces travel down this channel, when they find a control DNA with complementary bps, they anneal to form double stranded DNA
6. This causes sanger sequencing reaction to occur at the site of double stranded DNA. All of these reactions occur in parallel.
7. This is then read and recorded.

Question 8

What are the 2 methods of 2nd Generation sequencing

Answer 8

We have single end sequencing where we read the DNA sequence from just one end and then there’s the paired end sequencing where we read the sequencing from both ends

Question 9

What are the 2 different places at the DNA where we can do 2nd generation sequencing

Answer 9

1. Exome sequencing with the help of PCR or DNA oligos

2. Whole-genome sequencing

Question 10

What is the reference genome used

Answer 10

It is called haploid mosaic genome

Question 11

What is the controversy regarding using a reference genome

Answer 11

A reference genome can be significantly different for people of different ethnicities. Common and rare SNPs vary from population to population

Question 12

Pictorial difference in the exome sequencing and whole genome sequencing

Answer 12

In exome sequencing we only amplify the region around that gene

Question 13

What is 2nd generation RNA sequencing

Answer 13

We obtain DNA from reverse transcription of an RNA and then sequence that DNA to read the genome

Question 14

How can their be errors in gene alignment

Answer 14

The softwares we use for reading genomes aligns genes with the reference haploid mosaic genome being used. There are some variation in the genomic alignment as it is difficult to align at

1. Repetitive regions
2. Low complexity sequences
3. This presents a challenge to identify duplications (and maybe repetitions)