Part 1/2: Tools of Molecular Genetics

Question 1

Restriction enzymes:

Answer 1

Cleave DNA at specific sites.

• bacterial enzymes (nucleases)
• can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs (palindrome)
• Different restriction nucleases cut at different sequences.
• Extensively used in recombinant DNA technology.

Question 2

What types of DNA sequences do restriction enzymes cleave?

Answer 2

Palindromic

(display rotational symmetry)

Question 3

The two ways restriction enzymes can cleave palindromic sequences:

Answer 3

1. blunt ends (cut through center of sequence)
2. Sticky ends (uneven cuts)

Question 4

DNA/RNA Gel electrophoresis:

Answer 4

Separates DNA/RNA fragments by size.

• smaller strands move further down the gel
• visualized with ethidium bromide (flouresces) or autoradiography

Question 5

Hybridization:

Answer 5

Detects specific nucleotide sequences (DNA/RNA).

• Experimental process in which 2 complementary nucleic acid strands form a duplex
• uses DNA probe
• DNA/RNA double strand first denatured using high pH or temperature; denaturant removed to allow re-annealing
  
  • “stringency of conditions”

Question 6

Hybridization: how do you raise the stringency of re-annealing?

Answer 6

1. high stringency hybridization
   
   • increase the temperature or concentration of denaturing agent (will allow more perfect probe/strand annealing to occur)
2. use shorter probes

Question 7

Hybridization: effect of reduced stringency

Answer 7

• lower temperature and pH
• allows cross-species analyses and identification of distantly related members of a gene family
• use longer probe

Question 8

Specimen type analyzed via Southern Blots:

Answer 8

DNA

Question 9

Specimen type analyzed via Northern Blots:

Answer 9

RNA

Question 10

Specimen type analyzed via Western Blots:

Answer 10

Protein

Question 11

Restriction fragment length polymorphism (RFLP) steps:

Answer 11

Detects when the length of a detected fragment varies between individuals.

1. DNA fragmented by a restriction enzyme.
2. DNA fragments separated by length via gel electrophoresis.
3. DNA on gel transferred to a membrane (nitrocellulose/nylon paper).
4. Nitrocellulose/nylon paper with DNA fragments exposed to buffer with radioactive DNA probe.
5. Paper washed, unlabeled DNA washes off.
6. Autoradiography demonstrates DNA that binded to probe.

Question 12

An RFLP occurs when:

Answer 12

• the length of a detected fragment varies between individuals.
  
  • can be used in genetic analyses

Question 13

Northern Blotting:

Answer 13

Determines gene expression.

• used to determine the molecular weight of an mRNA and to measure relative amounts of the mRNA present in different samples.

Question 14

Northern Blotting Steps:

Answer 14

• run RNA fragments on gel to separate by length
• transfer to paper membrane using UV light
• hybridize membrane with radioactive probe

Question 15

DNA Microarray:

Answer 15

Anayzes expression of many genes at once.

• A glass slide upon which a large number of short DNA molecules (typically in the tens and thousands) have been immobilized in an orderly pattern.
• Each of these DNA fragments acts as a probe for a specific gene.

Question 16

DNA Microarray Steps:

Answer 16

1. mRNA from sample obtained.
2. mRNA transcribed to cDNA via reverse transcriptase.
3. Above samples put on glass slide upon which a large number of short DNA molecules (typically in the tens and thousands) have been immobilized in an orderly pattern.
4. Hybridization occurs.
5. Gene expression/overlap determined via flourescent microscope.

Question 17

Allele Specific Oligonucleotides (ASOs):

Answer 17

Detects a specific nucleotide sequence (allele).

• used to detect sickle cell and thallassemia mutations.

Question 18

PCR steps:

Answer 18

1. Denature DNA (heat)
2. Primer Annealing (cooling - hybridize)
3. Extension (DNA polymerase/free nucleotides)
4. Repeat

NEEDS HEAT-STABLE DNA POLYMERASE

Question 19

Polymerase chain reaction (PCR):

Answer 19

Amplifies specific regions of DNA.

• Technique for amplifying specific regions of DNA by a brief heat treatment to separate complementary DNA strands.
• Can amplify trace amounts of DNA.

Question 20

Allele Specific PCR:

Answer 20

Amplifies allele-specific DNA sequences.

• uses allele specific primers that perfectly basepair to the 3’ end nucleotide of a sequence

Question 21

Primers:

Answer 21

• small synthetic segments of single-stranded DNA
  
  • bind to a specific region on either side of the target DNA sequence and initiates replication of the target DNA at that point.
• specify the DNA sequence to be amplified

Question 22

Reverse Trancriptase PCR (RT-PCR)

Answer 22

Used to reverse-transcribe and amplify RNA to cDNA. Detects gene expression.

1. single stranded mRNA obtained from tissue
2. reverse transriptase transcribes mRNA to cDNA
3. RNAase removes RNA primer
4. PCR carried out
   
   1. denature
   2. anneal primer
   3. extend
   4. repeat

Question 23

What is required for Sanger Sequencing?

Answer 23

• single-stranded DNA template
• DNA primer
• DNA polymerase
• normal deoxynucleosidetriphosphates (dNTPs),
• modified di-deoxynucleosidetriphosphates (ddNTPs)
  
  • these terminate DNA strand elongation

Question 24

Sanger Sequencing Steps:

Answer 24

Used to sequence DNA in a stepwise fashion.

1. four individual reactions
2. target DNA strand replicated in presence of primer, polymerase, regular dNTPs, and one of the ddNTPs (A, G, T, or C).
3. Ran on a denaturing gel, separates fragments varying by one nucleotide.
4. Gel read from bottom (5’) to the top (3’). Determines DNA sequence.

Question 25

On a denaturing gel ran following Sanger Sequencing, what is the DNA sequence from the bottom of the gel to the top of the gel?

Answer 25

5’ → 3’

Question 26

Automated DNA sequencing:

Answer 26

Used to determine DNA sequence.

• a single reaction tube with target DNA, primer, dNTPs, and all four ddNTPs (A, G, C, and T).
• Each ddNTP is labeled with a different color flourescent.
• Separated by capillary gel electrophoresis.
• Laser detects flourescence, chromatogram generated by computer.
  
  • READ LEFT TO RIGHT

Question 27

Sanger Seguencing gels are read from:

Answer 27

• bottom to top (5’ → 3’)

Question 28

Automated DNA sequencing chromatograms are read from:

Answer 28

• left to right (5’ → 3’)

Question 29

Complementary DNA (cDNA):

Answer 29

• DNA molecules made as a copy of mRNA and therefore lacking the introns that are present in the genomic DNA.
• Used to determine the amino acid sequence of a protein by DNA sequencing or to make the protein in large quantities by cloning followed by expression.

Question 30

Dideoxy-DNA sequencing:

Answer 30

• The standard method of DNA sequencing.
• utilizes DNA polymerase and chain terminating nucleotides.
  
  • missing 3’ OH group

Question 31

Probe:

Answer 31

• A labeled segment of DNA or RNA used to find a specific sequence of nucleotides in a DNA molecule.
• May be synthesized in the laboratory, with a sequence complementary to the target DNA sequence.