Recombinant DNA And Biotechonology 11-17

Question 1

Primer design and construction

Answer 1

We need to know the sequence of two small flanking regions of the sequence we wish to amplify

Question 2

Denaturing the target protein in PCR

Answer 2

Produce single stranded DNA (will allow the primers to bind when cooled
-heat is used

Question 3

What is the primer for PCR?

Answer 3

DNA

Question 4

Annealing primers in PCR

Answer 4

DNA sample is cooled and the primers bind to the complementary sequence in the target DNA

Question 5

Chain extension in PCR

Answer 5

A DNA polymerase uses dNTPs to extend the primer and build a complementary copy of the DNA strand

Question 6

Steps _______ repeated 20-30 times (denature, anneal, extend)

Answer 6

2-4

Question 7

Why do you need to use heat stable Taq DNA polymerases?

Answer 7

So its stable at higher temps

Question 8

Advantages of PCR

Answer 8

• sensitivity
• speed
• DNA can be used for gel electrophoresis, southern blotting, etc
• mutation detection, detection of latent viruses, forensics, prenatal genetic diagnosis

Question 9

Northern blot

Answer 9

• mRNA
• quantitative AND qualitative
• expression of gene
• only one gene at a time

Question 10

Micro arrays

Answer 10

• mRNA levels
• contain thousands of immobilized sequences (probes) on glass slides
• compare global gene expression changes in different cell types
• synthesize and label cDNA from two different cell types with different fluorescent probes
• computer analyzed

Question 11

Proteins-proteomics

Answer 11

Similar to genomics but looks at all the different proteins produced in particular cell or tissue types (including post-translational modifications, enzyme modulation by phosphorylation , etc..
-more for research, not clinic

Question 12

ELISA (enzyme linked immunosorbent assays)

Answer 12

• antigen bound to the well of a microtiter
• probed with an antibody linked to an enzyme
• detect by adding substrate for enzyme to form a colored reaction

Question 13

False positive for ELISA

Answer 13

Sometimes you pick up a false positive

• if negative, you can feel confident about results
• if positive, follow up to make sure its not false positive

Question 14

Western blot

Answer 14

• proteins
• samples separated by gel electrophoresis based on size
• proteins blotted onto membrane
• probed with an enzyme-linked antibody to identify bands
• more specific than ELISA, but more time and labor intensive, less sensitive

Question 15

Analysis of DNA

Answer 15

• southern blot
• ASO
• PCR

Question 16

analysis of RNA

Answer 16

• northern blot
• microarray
• PCR

Question 17

Analysis of protein

Answer 17

• western blot
• ELISA
• proteomics

Question 18

Purpose of southern blot

Answer 18

Detects DNA changes

Question 19

Purpose of northern blot

Answer 19

Measures mRNA amounts

Question 20

Purpose of western blot

Answer 20

Measures protein amounts

Question 21

Purpose of ASO

Answer 21

Detects DNA mutations

Question 22

Purpose of microarray

Answer 22

Measures many mRNA levels at once

Question 23

Purpose of ELISA

Answer 23

Detects proteins (antigens) or antibodies

Question 24

Purpose of proteomics

Answer 24

Measures abundance, distribution, posttranslational modifications, functions, and interactions of cellular proteins

Question 25

Quantitative techniques

Answer 25

- northern blot - western blot - microarray - ELISA - proteomics - sometimes PCR

Question 26

Is DNA quantitative?

Answer 26

No

Question 27

Sickle cell mutation

Answer 27

B-globin gene that eliminates restriction site for restriction enzyme

Question 28

What does sickle cell create?

Answer 28

An RFLP: in this case, the disease causing mutation creates an RFLP that can be used for diagnosis (one larger fragment of DNA)

Question 29

In most diseases, an RLFP used for diagnosis ones what?

Answer 29

Is linked to the disease, but no the actual disease causing mutation

Question 30

RFLP in sickle cell

Answer 30

Enough DNA moving around you usually carry passenger polymorphism during crossover, not the case for sickle cell

Question 31

Restriction enzyme for sickle cell

Answer 31

Abolished

Question 32

Sickle cell and southern blot

Answer 32

With probe to beat-globin gene

Question 33

Normal cells vs sickle cells on southern blot

Answer 33

- normal is smaller band | - sickle cell is larger banded

Question 34

Genetic testing for sickle cell: alternative

Answer 34

Could do a similar procedure using PCR, amplify the region containing the potential mutation, digest PCR products with MstII

Question 35

PCR for sickle cell

Answer 35

- use primers flanking beta globin gene, or region where mutation may be - digest PCR product with MSTII, run on gel - larger band for sickle cell

Question 36

ASO and sickle cells

Answer 36

ASO probes-Allen specific oligonucleotides VERY SPECIFIC -very reliable and specific, very fast, minimal complications with the technique, non-invasive, inexpensive