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BIOCHEM (1st yr Med) - Harper's > 39 - Recombinant DNA technology > Flashcards

39 - Recombinant DNA technology Flashcards

1
Q

Manipulation of a DNA sequence and the construction of chimeric molecules

A

Genetic Engineering

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2
Q

The combination of genetic material of 2 or more organisms

i.e. Human DNA + bacterial DNA

A

Chimeric Molecules

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3
Q

DNA Review:

What are exons?

A
  • Coding region
  • remains in the mature mRNA after processing of primary transcript
  • shorter sequences
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4
Q

DNA Review:

What are introns?

A
  • non-coding region
  • are the ones removed during the processing of the primary transcript
  • in between exons
  • not present in mRNA
  • longer sequences
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5
Q

(True/False)

Introns are always present in mature mRNA

A

False

During processing, introns are removed and exons are the ones that compose the mature mRNA

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6
Q

The process of removing the introns in the primary transcript before it is transported to the cytoplasm

A

RNA splicing

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7
Q

What is the 5’ Flanking Sequence DNA?

A
  • found before the transcription start site
  • contains regulatory elements (i.e. promoters, enhancers, silencer)
  • determine WHEN and HOW MUCH primary transcript are produced > Gene Expression
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8
Q

The region that precedes the starting point of a gene, also known as the transcription start site. This sequence is typically located on the 5’ end of the DNA strand.

A

5’ flanking sequence DNA

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9
Q

Definition:

Promoter regions

A

Specific sequences of DNA that are recognized by RNA polymerase, the enzyme that synthesizes the mRNA transcript.

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10
Q

Definition:

Enhancers

A

Regulatory elements that can increase the transcriptional activity of the promoter.

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11
Q

Definition:

Silencers

A

Regulatory elements that decrease transcriptional activity.

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12
Q

Sequences of DNA that control the transcription of a gene by determining when and how much mRNA is produced.

A

Regulatory elements

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13
Q

The process by which the expression of a gene is controlled, typically through the activity of regulatory elements and proteins.

A

Gene Regulation

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14
Q

Review:

DNA Structure

A
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15
Q

Review:

DNA Structure -Bonds

A
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16
Q

Review:

Nucleic Acid Structure

A
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17
Q

Type of DNA preparation where mRNA as the sample is converted to DNA

Enzyme reverse transcriptase is used

A

copy or complimentary DNA
cDNA

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18
Q

Type of DNA preparation that digest extracted DNA with a restriction enzyme

A

Genomic DNA

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19
Q

A restriction enzyme that cut the DNA at specific DNA sequence within the molecule

A

Endonucleases

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20
Q

A restriction enzyme that digest from the ends of DNA molecule

A

Exonucleases

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21
Q

Enumerate the types of restriction enzymes

A
  1. endonuclease
  2. exonuclease
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22
Q

A single-stranded DNA molecule used to detect complementary DNA sequences.

Can be labeled with detectable markers for visualization and quantification

A

DNA probe

Hybridization is used to bind the probe to a sample containing the complementary sequence.

The probe and target DNA form a stable hybrid that can be detected and quantified

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23
Q

From DNA it makes complimentary DNA copies

Used in:
1) creating DNA probes
2) DNA amplification
3) DNA Sequencing

A

DNA-dependent Polymerase

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24
Q

Derived from RNA tumor viruses that make cDNA copies of RNA templates

Useful in cloning sequences that are complimentary to mRNA

A

RNA-dependent polymerase

Useful in cloning sequences that are complimentary to mRNA

25
Q

Restriction enzyme origin

A

Found in Bacterial genes

used to protect short bacterial DNA from infective phages that try to attach themselves to the DNA for proliferation

26
Q

An organism with a restrictive enzyme will always have

A

site specific DNA methylases

Restrictive enzymes cut DNA at specific sequences. If a bacteria has a restrictive enzyme for XXX sequence to protect from viruses with XXX sequence, the bacteria will have DNA methylases to protect its own XXX sequence from its own restrictive enzyme

27
Q

Site specific DNA methylates

A

Enzymes that methylates host DNA

28
Q

Sticky ends

A

specific DNA sequence that forms after DNA is digested by restrictive enzymes

useful in constructing hybrid or chimeric DNA molecules

29
Q

Characteristic linear array of sites for various enzymes

A

Restriction map

30
Q
  • Result from cutting both strands of DNA in the same position
  • No overhanging nucleotides (homopolymer tailing)
  • Fragments cannot base pair with other blunt ends
  • Difficult to join together without additional enzymes or techniques use enzyme: terminal transferase
A

Blunt ends

31
Q
  • Result from cutting DNA in a staggered fashion
  • Overhanging single-stranded DNA sequences at each end
  • Fragments have complementary overhangs that can base pair with each other
  • Can be easily joined together using DNA ligase
  • Commonly used in recombinant DNA technology
A

Sticky ends

Formed by endonucleases that cleaves a specific double-stranded DNA sequence (4-7 bp long)

32
Q

Seals overhangs to make recombinant DNA by catalyzing the formation of phosphodiester bond between adjacent 3’-hydroxyl and 5’ -phosphate ends of DNA

A

DNA ligase

33
Q

Carrier of target DNA

A

Vector

34
Q

Essential features of Vectors

A
  • able to replicate
  • capable of insertion of DNA of interest
  • selectable marker
  • contain a site for insertion of target DNA
35
Q

Extrachromosomal circular pieces of DNA found in bacteria

Can replicate within the host bacterium

A

Plasmids

Caryy genes that code for proteins that confer antibiotic resistance to the host

Type of vector (?)

36
Q

Small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance

A

Plasmids

37
Q

Viruses that infect bacteria

can be genetically modified to produce cloning vectors
- more efficient in transecting the host
- can hold a bigger fragment of DNA
- linar DNA

A

Bacteriophages

38
Q

Plasmids that contain DNA sequences (cos sites) required for packaging lambda DNA into package particles

Is a DNA cloning vector that combines the best features of plasmids and phages

A

cosmid

39
Q

Accept 1000kb inserts and are easily propagated in yeast cells

Helpful in studying Human genome

A

YAC or Yeast Artifical Chromosome

40
Q

Vectors SUMMARY

A
41
Q

Identical host cells that carry an identical recombinant DNA molecule

A

Clones

42
Q

Basic cloning strategy

A

1) Recombinant DNA is made (from vector and target DNA by annealing and ligation)
2) Recombinant DNA is introduced to the host via transformation or transfection
3) propagation in host bacterial cells
4) target DNA is specifically selected by hybridization

43
Q

Gene cloning steps

A

1) vector and insert DNA are digested with the same restricion enzyme (ends of insert DNA and vector DNA match)
2) restriction digests of vector and insert DNA are mixed and allowed to reanneal with DNA ligase
3) vecrtor is propagated in bacteria
4) target DNA selected by various detection procedure

44
Q

Blotting Techniques

A

“SNoW DRoP”

Southern Blotting - DNA
Northern Blotting - RNA
Western Blotting - Protein

45
Q

Can show overexpression of oncogenes and down regulation of tumor-suppressor genes in cancerous cells

Used in gene expression in the rejection of transplanted organs

A

Northern Blot

46
Q

Used to determine whether a transgenic organism expresses the inserted gene to produce a protein

A

Western Blotting

47
Q

Used for prenatal diagnosis

A

Southern Blot

48
Q

Allows one to observe a particular gene’s expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, etc.

A

Northern Blot

49
Q

Uses labelled antibodies to detect proteins

A

Western Blot

50
Q

Chemical DNA sequencing by

A

Maxam & Gilbert

51
Q

Enzymatical DNA sequencing by

A

Sanger

52
Q

(True/False)

The chemical DNA sequencing procedure is easier and quantitatively more superior than the enzymatic procedure

A

False

53
Q

Used for DNA amplification

A

Polymerase Chain Reaction (PCR)

54
Q

RTPCR

A

Reverse Transcription Polymerase Chain Reaction

In RT-PCR, RNA molecules are first converted to complementary DNA (cDNA) using the enzyme reverse transcriptase.

The cDNA is then amplified using the polymerase chain reaction (PCR) technique to produce millions of copies of the target RNA sequence.

55
Q

A variation of RFLP analysis

A

DNA Fingerprinting

56
Q

A method for detecting protein-DNA interactions by applying labeled DNA probe to a transfer membrane that contains renatured protein

A

Southwerstern Blot

57
Q

Comprises complementary DNA copies of the population of mRNAs in a tissue

A

cDNA Library

58
Q

What is a tool in the diagnosis of single base changes like sickle-cell disease?

A

Restriction Fragment Length Polymorphism

BIOCHEM (1st yr Med) - Harper's (13 decks)

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