Precision medicine

Question 1

How long is Human Genome. How many functional genes? What %?

Answer 1

Around 3B bp. 20000, 1.2%

Question 2

What is found at the linear end of chromosomes

Answer 2

Telomere

Question 3

What is found at the middle of chromosome

Answer 3

Centromere

Question 4

What is the nature of telomere and centromere

Answer 4

HIghly repetitive DNA sequences

Question 5

How do we name the subdivision of chromosomes

Answer 5

Cytobands

Question 6

What are the components of Gene

Answer 6

Introns, exons, regulatory element, promotor

Question 7

What are the 4 criterias for healthy gene

Answer 7

Organised, Stable,Copied and usable

Question 8

Does genetic variation affect most or some phenotypes that are observed including diseases

Answer 8

Most

Question 9

Name the 3 level of genetic variation and examples

Answer 9

1. Copy number varation

Like Down’s Syndrome

2. Structural variation
   Example: Deletion, insertion, Addition, translocation

e.g. FSHD (<11 copies of D4Z4, norm: 11-100)

3. Sequence level variation
   Like SIngle Nucleotide Polymorphism (SNP)

Example: Sickle cell disease: T–>Ａ. F –>Ｖ

Question 10

What is PCR stands for and it’s purpose

Answer 10

It is polymerase Chain Reaction to amplify the quantity of DNA

Question 11

Steps for PCR

Answer 11

1. Denature the DNA with high temperature to separate them into 2 strands
2. 2 primers should be added to the target site
3. The primer is extended with the use of Tag DNA polymerase and dNTP. phosphodiester bonds form between the 3’OH group of growing DNA and 5’ triphosphate group of dNTP, and the Tag removes the Beta and Gamma phosphate group from the recently added dNTP after formatiomn of hydrogen bond to let other join.

Question 12

Name 2 methods for gene sequencing

Answer 12

Sanger’s Method and NExt generation sequencing technique

Question 13

What are the procedures for Sanger’s Method

Answer 13

It involves

1. Add a primer onto the template DNA strand
2. Use DNA polymerase to add nucleotides onto it, until it randomly add a fluorescently labelled dideoxyribonucleotide
3. As the dideoxyribonucleotide cannot allow any more nucleotide to bind onto it, it act as the terminal for the DNA sequence.
4. It produces fragments of different sizes. It repeats until fragments of the all sizes are produced. The sequence is read from the colour

Question 14

What is the problem of Sanger’s method

Answer 14

VERY labour intensive for long sequence.

Question 15

What is the capability and base length for the Sanger’s method

Answer 15

1 at a time, 600-800 bp

Question 16

What are the procedures of Next-generation sequencing

Answer 16

1.In a first step, sequencing libraries are prepared from fragmented DNA samples. Adaptors and barcodes(for identification) are attached to these fragments which aid the attachment to the sequencing flow cell, the amplification reaction and the pooling of multiple samples into a single experiment.

2.Clustering: The DNA fragments are amplified on the solid surface of sequencing flow leading to clusters of identical sequences for better detection.

3.The four nucleotides carry unique fluorescent dyes and compete for addition to a growing DNA strand. Only the nucleotide complementary to the DNA template is incorporated.

4.Upon excitation of by a light source a base specific fluorescent signal is emitted allowing the identification of the base (base call).

5.Next, the fluorophores are cleaved off and removed which produces a regenerated 3’OH group permitting the addition of the next complementary nucleotides in the next sequencing cycle.

6.In this way billions of DNA fragments are sequenced in a massively parallel fashion. The last step is the analysis of the sequencing data which will depend on the specific application of the experiment.

Question 17

What is the range of short-reading NGS, accuracy and how many can be read simultaneously.

Answer 17

150-300 bp, 99%, millions

Question 18

Under what circumstances is NGS not that great

Answer 18

When have to cope with a lot ofdata as short sized DNA and PCR are involved