39 - Recombinant DNA technology

Question 1

Manipulation of a DNA sequence and the construction of chimeric molecules

Answer 1

Genetic Engineering

Question 2

The combination of genetic material of 2 or more organisms

i.e. Human DNA + bacterial DNA

Answer 2

Chimeric Molecules

Question 3

DNA Review:

What are exons?

Answer 3

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

Question 4

DNA Review:

What are introns?

Answer 4

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

Question 5

(True/False)

Introns are always present in mature mRNA

Answer 5

False

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

Question 6

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

Answer 6

RNA splicing

Question 7

What is the 5’ Flanking Sequence DNA?

Answer 7

• 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

Question 8

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.

Answer 8

5’ flanking sequence DNA

Question 9

Definition:

Promoter regions

Answer 9

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

Question 10

Definition:

Enhancers

Answer 10

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

Question 11

Definition:

Silencers

Answer 11

Regulatory elements that decrease transcriptional activity.

Question 12

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

Answer 12

Regulatory elements

Question 13

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

Answer 13

Gene Regulation

Question 14

Review:

DNA Structure

Answer 14

Question 15

Review:

DNA Structure -Bonds

Answer 15

Question 16

Review:

Nucleic Acid Structure

Answer 16

Question 17

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

Enzyme reverse transcriptase is used

Answer 17

copy or complimentary DNA
cDNA

Question 18

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

Answer 18

Genomic DNA

Question 19

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

Answer 19

Endonucleases

Question 20

A restriction enzyme that digest from the ends of DNA molecule

Answer 20

Exonucleases

Question 21

Enumerate the types of restriction enzymes

Answer 21

1. endonuclease
2. exonuclease

Question 22

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

Can be labeled with detectable markers for visualization and quantification

Answer 22

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

Question 23

From DNA it makes complimentary DNA copies

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

Answer 23

DNA-dependent Polymerase

Question 24

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

Useful in cloning sequences that are complimentary to mRNA

Answer 24

RNA-dependent polymerase

Useful in cloning sequences that are complimentary to mRNA

Question 25

Restriction enzyme origin

Answer 25

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

Question 26

An organism with a restrictive enzyme will always have

Answer 26

site specific DNA methylases ## Footnote 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

Question 27

Site specific DNA methylates

Answer 27

Enzymes that methylates host DNA

Question 28

Sticky ends

Answer 28

**specific** DNA sequence that forms after DNA is digested by restrictive enzymes ## Footnote useful in constructing hybrid or chimeric DNA molecules

Question 29

Characteristic linear array of sites for various enzymes

Answer 29

Restriction map

Question 30

- 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**

Answer 30

Blunt ends

Question 31

- 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

Answer 31

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

Question 32

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

Answer 32

DNA ligase

Question 33

Carrier of target DNA

Answer 33

Vector

Question 34

Essential features of Vectors

Answer 34

- able to replicate - capable of insertion of DNA of interest - selectable marker - contain a site for insertion of target DNA

Question 35

Extrachromosomal circular pieces of DNA found in bacteria Can replicate within the host bacterium

Answer 35

Plasmids ## Footnote Caryy genes that code for proteins that confer antibiotic resistance to the host Type of vector (?)

Question 36

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

Answer 36

Plasmids

Question 37

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

Answer 37

Bacteriophages

Question 38

Plasmids that contain DNA sequences (cos sites) required for packaging lambda DNA into package particles ## Footnote Is a DNA cloning vector that combines the best features of plasmids and phages

Answer 38

cosmid

Question 39

Accept 1000kb inserts and are easily propagated in yeast cells ## Footnote Helpful in studying Human genome

Answer 39

YAC or Yeast Artifical Chromosome

Question 40

Vectors SUMMARY

Answer 40

Question 41

Identical host cells that carry an identical recombinant DNA molecule

Answer 41

Clones

Question 42

Basic cloning strategy

Answer 42

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

Question 43

Gene cloning steps

Answer 43

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

Question 44

Blotting Techniques

Answer 44

"SNoW DRoP" Southern Blotting - DNA Northern Blotting - RNA Western Blotting - Protein

Question 45

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

Answer 45

Northern Blot

Question 46

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

Answer 46

Western Blotting

Question 47

Used for *prenatal diagnosis*

Answer 47

Southern Blot

Question 48

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

Answer 48

Northern Blot

Question 49

Uses labelled antibodies to detect proteins

Answer 49

Western Blot

Question 50

Chemical DNA sequencing by

Answer 50

Maxam & Gilbert

Question 51

Enzymatical DNA sequencing by

Answer 51

Sanger

Question 52

# (True/False) The chemical DNA sequencing procedure is easier and quantitatively more superior than the enzymatic procedure

Answer 52

False

Question 53

Used for DNA amplification

Answer 53

Polymerase Chain Reaction (PCR)

Question 54

RTPCR

Answer 54

Reverse Transcription Polymerase Chain Reaction ## Footnote 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.

Question 55

A variation of RFLP analysis

Answer 55

DNA Fingerprinting

Question 56

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

Answer 56

Southwerstern Blot

Question 57

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

Answer 57

cDNA Library

Question 58

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

Answer 58

Restriction Fragment Length Polymorphism