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Biochem BLOCK 4 > Recombinant DNA And Biotechnology 11-16 > Flashcards

Recombinant DNA And Biotechnology 11-16 Flashcards

1
Q

DNA from a cDNA library can be cloned into an ________ _______ for _______ of ___________

A

Expression vector

Production of proteins

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2
Q

Bacterial expression vector

A

-promoter, shine dalgarno sequence and cDNA are used to transform expression bacteria strains

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3
Q

Why use yeast as a vector?

A

Bacteria have no organelles

Yeast can express cell surface proteins

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4
Q

DNA sequencing is a technique used to determine…

A

The exact sequence of a cloned or PCR amplified stretch of DNA

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5
Q

How do you get a single stranded DNA template?

A

Melt it

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6
Q

What are major components of the reaction used to conduct sequencing

A

DNA + dNTPs + DNA primer + polymerase

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7
Q

What does each of the 4 tubes contain in DNA sequencing

A
  • ddCTP
  • ddATP
  • ddGTP
  • ddTTP
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8
Q

What stops elongation of DNA strand?

A

DdNTP

-used in cancer treatments

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9
Q

What are probes used for

A

Identify DNA fragments

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10
Q

What is a probe

A

A ssDNA molecule, labeled using radioactivity that be by hybridized a ssDNA that is complementary

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11
Q

Hybridization

A
  • DNA made single stranded
  • target immobilized on support so it wont reanneal
  • ssDNA coated membrane exposed to probe
  • if complentary seq Preston, probe will bind and can be identified via autoradiography
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12
Q

Smaller probes

A
  • chemically synthesized oligonucleotides (2-30 base pairs)
  • very specific
  • 1 nucleotide difference can affect recognition
  • DIAGNOSTIC
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13
Q

Larger probes

A
  • made via one of several molecular biology techniques
  • much less specific
  • heritage
  • COMPARATIVE
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14
Q

Which type of probe is diagnostic

A

Smaller

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15
Q

Which type of probe is comparative

A

Larger

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16
Q

Southern blotting

A
  • analysis of DNA
  • restriction digestion
  • gel electrophoresis
  • denatured and blotted
  • blot probed
17
Q

Northern blot

A
  • analysis of RNA (mRNA)
  • complentary mRNA
  • only detects expressed sequences
  • can be used for tissue or cell specific studies
  • QUANTITATIVE (to measure gene expression)
18
Q

Western blot

A
  • analysis of protein
  • probe is antibody specific to the protein of interest
  • quantitative
19
Q

What is the frequency of nucleotides that are different in humans who are not related

A

1 in 1500

20
Q

Genetic variations in non-coding regions with no disease

A

Polymorphisms

21
Q

The term ______ is usually reserved for genetic changes that cause disease

A

Mutation

22
Q

When is a RFLP present?

A

If a genetic change in a polymorphic region creates or deleted a restriction site OR has more or less of a repeated sequence

23
Q

DNA variations resulting in RFLPS

A
  1. Single nucleotide polymorphisms (SNPs)

- variable number or tandem repeats (VNTRs)

24
Q

Single nucleotide polymorphisms (SNPs)

A
  • single nucleotide change
  • 90% of genetic variation
  • may create or abolish restriction site***
  • occasionally an RFLP is created by a disease causing mutation
  • more often it is a harmless change that results in a different restriction pattern
25
Q

Variable number of tandem repeats (VNTRs)

A
  • human genome contains many regions where a sequence is repeated in tandem many times
  • varies from person to person and unique for individual
  • if DNA is cleaved on either side of a VNTR, an RFLP is produced
26
Q

RFLPs based on SNPs

A
  • creation of abolishment of a restriction site
  • different size RFLPs based on different restriction cutting
  • used to mark genes (alleles), disease markers
27
Q

RFLPs based on VNTRs

A
  • more or less of a tandem repeat
  • different size RFLPs based on different number of repeats
  • used as molecular fingerprints (paternity tests)
28
Q

DNA cloning

A

Amplify fragments by inserting them into a vector and the host replicating the vector

29
Q

PCR

A

Amplification done completely in a tube

Biochem BLOCK 4 (11 decks)

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