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Biochem BLOCK 4 > Recombinant DNA And Biotechonology 11-17 > Flashcards

Recombinant DNA And Biotechonology 11-17 Flashcards

1
Q

Primer design and construction

A

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

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2
Q

Denaturing the target protein in PCR

A

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

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3
Q

What is the primer for PCR?

A

DNA

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4
Q

Annealing primers in PCR

A

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

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5
Q

Chain extension in PCR

A

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

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6
Q

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

A

2-4

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7
Q

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

A

So its stable at higher temps

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8
Q

Advantages of PCR

A
  • sensitivity
  • speed
  • DNA can be used for gel electrophoresis, southern blotting, etc
  • mutation detection, detection of latent viruses, forensics, prenatal genetic diagnosis
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9
Q

Northern blot

A
  • mRNA
  • quantitative AND qualitative
  • expression of gene
  • only one gene at a time
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10
Q

Micro arrays

A
  • 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
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11
Q

Proteins-proteomics

A

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

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12
Q

ELISA (enzyme linked immunosorbent assays)

A
  • 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
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13
Q

False positive for ELISA

A

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
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14
Q

Western blot

A
  • 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
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15
Q

Analysis of DNA

A
  • southern blot
  • ASO
  • PCR
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16
Q

analysis of RNA

A
  • northern blot
  • microarray
  • PCR
17
Q

Analysis of protein

A
  • western blot
  • ELISA
  • proteomics
18
Q

Purpose of southern blot

A

Detects DNA changes

19
Q

Purpose of northern blot

A

Measures mRNA amounts

20
Q

Purpose of western blot

A

Measures protein amounts

21
Q

Purpose of ASO

A

Detects DNA mutations

22
Q

Purpose of microarray

A

Measures many mRNA levels at once

23
Q

Purpose of ELISA

A

Detects proteins (antigens) or antibodies

24
Q

Purpose of proteomics

A

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

25
Q

Quantitative techniques

A
  • northern blot
  • western blot
  • microarray
  • ELISA
  • proteomics
  • sometimes PCR
26
Q

Is DNA quantitative?

A

No

27
Q

Sickle cell mutation

A

B-globin gene that eliminates restriction site for restriction enzyme

28
Q

What does sickle cell create?

A

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

29
Q

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

A

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

30
Q

RFLP in sickle cell

A

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

31
Q

Restriction enzyme for sickle cell

A

Abolished

32
Q

Sickle cell and southern blot

A

With probe to beat-globin gene

33
Q

Normal cells vs sickle cells on southern blot

A
  • normal is smaller band

- sickle cell is larger banded

34
Q

Genetic testing for sickle cell: alternative

A

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

35
Q

PCR for sickle cell

A
  • 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
36
Q

ASO and sickle cells

A

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

Biochem BLOCK 4 (11 decks)

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