Chapter 19 - Molecular Genetics and Biotechnology

Question 1

What are the 4 key innovations in molecular genetics?

Answer 1

Recombinant DNA technology

Polymerase chain reaction

DNA sequencing

Genome editing systems

Question 2

What are the 6 challenges with working at the molecular level?

Answer 2

Genes are minute

Small physical quantities

Stability of molecules

Complexity of genome (introns, etc.)

Single cell has millions of genes

Nucleotides can’t be seen

Question 3

What is recombinant DNA technology?

Answer 3

AKA: genetic engineering

Take a segment of DNA and transfer it to another cell to study it

Allows us to locate, isolate, alter, combine, and study DNA fragments/genes

Question 4

What does recombinant mean?

Answer 4

Taking DNA from two sources and putting them together

Question 5

What are restriction enzymes and what is their role in recombinant DNA technology?

Answer 5

Role: Isolate DNA sequence

• Recognize specific nucleotide sequences and make double-stranded cuts at the specific restriction sites
• Produced by most eukaryotes
• 4-8 base pair cutters
• Sticky ends: have overhangs of nucleotides
• Blunt ends: no overhang
• DNA molecules cut with the same restriction enzyme have complementary sticky ends that pair if fragments are mixed together
• Shorter restriction enzymes may have a sequence that is seen more throughout the genome, which makes it cut more

Question 6

What are the 3 parts of recombinant DNA technology?

Answer 6

Restriction enzymes - isolate DNA sequence

Visualizing DNA

Cloning vectors - DNA inserted into new source

Question 7

What are the 2 methods for visualizing DNA in recombinant DNA technology?

Answer 7

Gel electrophoresis

Blotting

Question 8

What is the process for gel electrophoresis?

Answer 8

DNA passed through gel matrix, which allows certain sizes of DNA or nucleic acids or proteins to pass through it in a size manner

Charged current is passed from negative to positive because DNA is negatively charged

Gel is stained with ethidium bromide (intercalating agent)

Gel put under UV light and DNA bands fluoresce

Probes can locate fragments
- Can stain certain fragments to identify them

Can run DNA, RNA, and proteins through gels

Question 9

What is the process of blotting and its 3 types?

Answer 9

Gels can be transferred to a solid matrix
- Probe then used to stain molecules on membrane

Types:
- Western – proteins
- Southern – RNA
- Northern – DNA

Question 10

What are cloning vectors and why is it used in recombinant DNA technology?

Answer 10

Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into another cell

Putting gene into new source, which allows it to freely replicate and clone itself in new source

Typically plasmid vectors - circular DNA molecules that exist naturally in bacteria

Question 11

What are the 3 characteristics of a cloning vector?

Answer 11

Origin of replication

Selectable markers – enables any cells containing vector to be identified and selected for

One or more unique restriction site into which the DNA fragment can be inserted
- Fracture vector too heavily if you have more than one restriction site

Question 12

What are the steps to combining and taking up a cloning vector?

Answer 12

Combining:
- Cut foreign DNA to isolate it, and run it on a gel to confirm/identify it
- Cut plasmid vector at restriction site
- Take foreign DNA sticky ends and join them to the sticky end complements on the plasmid vector DNA
- Sealed by DNA ligase

Taking up:
- Put plasmid vector into medium
- Grow bacteria (likely E. coli) in medium
- Plasmid vector taken up through transformation
- Plasmid vector can freely replicate with foreign DNA

Question 13

What test is done to know which bacteria will take up a plasmid vector with a gene fragment, and how does it work?

Answer 13

Blue-white screen

Selectable marker is an antibiotic resistant marker that confers antibiotic resistance to E. coli that has taken up plasmid vector
- Any bacteria that doesn’t have plasmid vector (whether it has gene fragment or not) will not grow on blue-white medium

Use gene disruption to determine if gene fragment is present
- Utilize plasmid vector that has a partial lacZ gene (codes beta-galactosidase)
- Restriction site sits inside partial lacZ gene
- If we cut lacZ gene to add gene fragment, the gene will be disrupted
- If lacZ is cut but no fragment is added, it will reseal itself together and will be intact

Question 14

What are the steps for a blue-white screen?

Answer 14

Grow bacteria on ampicillin and X-gal medium
- Ampicillin selects only antibiotic resistant bacteria (those with selectable marker)

Bacteria with an intact lacZ (lacZ+) gene in plasmid can cleave X-gal
- Produce blue pigment in colonies when they cleave X-gal

Bacteria with disrupted lacZ (lacZ-) cannot cleave X-gal because they don’t have beta-galactosidase
- Produce white pigment in colonies

Pick out white colonies because we only want bacteria with gene fragment in plasmid vector

Question 15

IGNORE
What are the 4 other types of cloning vectors and when are they used?

Answer 15

Phage-lambda vectors – viral vectors that infect E. coli at a high rate

Cosmids

Bacterial artificial chromosomes (bac) – can hold larger fragments

Expression vectors – replicate gene as well as protein that gene encodes
- Has sequences necessary for both transcription and translation

Used with larger fragments - the larger the fragment, the worse plasmid vectors are

Question 16

What are the applications of recombinant DNA technology?

Answer 16

Crops/plants
- Genetically modified plants that have herbicide, pesticide, and drought resistance tend to utilize recombinant DNA technology

Vectors that can be utilized to transfer bacterial genes into eukaryotic cells – called yeast artificial chromosomes (yac)
- Can also use retroviral vectors

Question 17

How is DNA amplified?

Answer 17

Polymerase Chain Reaction (PCR)

Allows for DNA to be exponentially replicated within a few hours

Can do this from very small amounts of DNA

Question 18

What are the steps of PCR?

Answer 18

Denaturing – DNA is heated up and pulled apart into two single strands

Annealing – primers stuck onto two template strands that specify fragment want to amplify on

Extension – new DNA fragments synthesized by DNA polymerase and dNTPs
- Specific polymerase used is Taq polymerase

Cycle through steps about 30 times to replicate DNA exponentially

Question 19

What are the 4 limitations to PCR?

Answer 19

Have to have prior knowledge of desired DNA sequence to be able to amplify it

If there’s contamination, the primer may not be specific enough to get the right sequence

Taq is not very accurate because it is fast and lacks proofreading capabilities
- Can alter polymerases to create higher-fidelity polymerases, which decrease error rate by introducing proofreading capabilities

Taq can only replicate small fragments ~2000-2500 base pairs
- Can overcome this by increasing Taq’s fidelity to create longer strands
- Can also modify other reaction conditions to increase the efficiency of Taq

Question 20

What are the 3 applications of PCR?

Answer 20

Can detect viral DNA in isolates

Can located rare, secretive organisms in an environment

Can detect genetic variation in populations

Question 21

What is the purpose of sequencing DNA and what are the 5 types?

Answer 21

Determine sequence of bases in a DNA molecule

Types:
- Dideoxy sequencing (Sanger sequencing)
- Sequencing in parallel
- Sequencing by synthesis
- Third generation sequencing
- DNA fingerprinting

Question 22

What is the basic idea of dideoxy/Sanger sequencing?

Answer 22

Take PCR product (amplified DNA) and use dye terminators (ddNTPs) that lack 3’-OH group that dNTPs have

Question 23

What is the process for old Sanger sequencing?

Answer 23

Take gene template and do another PCR with ddNTPs instead of regular dNTPs

When ddNTPs get incorporated into new strand, synthesis stops because the ddNTP lacks 3’-OH

Synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a ddNTP with the same base

The fragments produced in each reaction are separated by gel electrophoresis

The sequence can be read directly from the bands that appear on the autoradiograph of the gel, starting from the bottom

The sequence obtained is the complement of the original strand

Question 24

What is the process for current Sanger sequencing?

Answer 24

Each of the 4 ddNTPs is tagged with a different fluorescent dye, and the Sanger sequencing reaction is carried out

Fragments that end in the same base have the same colored dye attached

The products are denatured, and the DNA fragments produced by the reaction are loaded into a single well on an electrophoresis gel

Fragments migrate through gel according to size and the fluorescent dye on the DNA is detected by a laser beam

Each fragment appears as a peak on the computer printout – color of peak indicates which base is present

Question 25

What is the limitation of Sanger sequencing?

Answer 25

Since it’s based on PCR, can only get small gene sequences

Question 26

What is sequencing in parallel?

Answer 26

Millions of DNA fragments sequenced all at once

Question 27

What is the process for sequencing by synthesis?

Answer 27

DNA is chopped up into small fragments, which end up getting washed across a flow cell in a sequencer, where there are places where fragments can get stuck and attach to flow cell

Primers and polymerases are added to flow cell and the complementary strand of the fragments are synthesized

As synthesis occurs, fluorescent nucleotides are added in and are read by a computer, which reads every nucleotide and what color it fluoresces

After it fluoresces, it is pulled off and a non-fluorescent is added, and then the next fluorescent is added

Can have millions of nucleotides being read on flow cell all at one time

Question 28

What are the 2 types of third generation sequencing?

Answer 28

PacBio:
- Very accurate and long read lengths
- Typically reads 10,000-200,000 base pairs
- Relatively time consuming
- Tends to be expensive

Oxford nanophore
- Higher error rate, but still long read lengths
- Very small (size of a USB drive), so sequencing can be done in the field
- As DNA passes through nanophore, it disrupts an electrical current in the membrane
- A computer reads the change in current as the base sequence

Question 29

How does DNA fingerprinting work?

Answer 29

Can identify individuals based on unique genetic markers

Uses microsatellites/short tandem repeats (STRs) found all over the genome
- Number of repeats seen is unique between individuals

Can identify STRs using PCR with primers that sit on either side of STRs

The more STRs, the longer the sequence

Can amplify several STR regions in an individual
- Probability of the same STRs between individuals is very low, especially when amplifying several regions

Homozygotes for an allele will have 1 large peak

Heterozygotes for an allele will have 2 smaller peaks

Can also tell parentage and use in crime scenes because of uniqueness

Question 30

What is the main form of gene editing used?

Answer 30

CRISPR-Cas9

Question 31

How does CRISPR-Cas9 work?

Answer 31

Naturally occurring in bacteria and used for immunity and protection against viruses and other invading DNA elements
- CRISPR array consists of palindromic sequence and spacers (sequences from phages or other invaders)
- When a plasmid or phage enters cells, protein cuts up foreign DNA and inserts it into CRISPR array, which is transcribed into precursor RNA that is cleaved into smaller CRISPR-RNA
- CRISPR-RNAs combine with Cas proteins to form effector complexes
- When invading DNA comes in, it finds effector complex and cleaves it to stop it from replicating in host cell

Can be engineered to find unique sequences in the genome for editing
- Can use 2 RNA molecules – CRISPR-RNA and tracr-RNA that combine with CAS9 to form effector complex
- Can then be used to cleave specific DNA sites
- Single guide RNA (sgRNA) can help find genome we want to edit (Seed sequence)
- Can be modified to whatever sequence you want to edit

Question 32

What are the 2 advantages to CRISPR-Cas9?

Answer 32

Long seed sequences

Applied to many different organisms

Question 33

What are the 4 disadvantages to CRISPR-Cas9?

Answer 33

Off-site cleavage – cleavage in the wrong place

Can cause genetic mosaics – only alter some cells

Can’t always deliver all components into all cells

A lot of ethics associated

Question 34

What are the 3 methods of controlling gene function?

Answer 34

Forward genetics

Reverse genetics

RNAi

Question 35

What is forward genetics?

Answer 35

Begins with a phenotype (a mutant individual) and proceeds to a gene that encodes the phenotype

Question 36

What is reverse genetics?

Answer 36

Begin with a specific genotype, alter or inhibit it in some way, and determine phenotype from it

Types:
- Site-directed mutagenesis
- Transgenics and knock-outs

Question 37

What is site-directed mutagenesis?

Answer 37

Cuts out a short sequence of nucleotides with restriction enzymes and then replaces it with a synthetic oligonucleotide that contains the desired mutated sequence

Question 38

What are transgenics and knockouts?

Answer 38

Transgenics: DNA sequence of interest added to a genome that normally lacks it, then effect of introduced sequence on phenotype is observed

Knockouts: DNA sequence of interest is disabled in a genome and the phenotype is analyzed as it gives a good indication of the function of the missing gene

Question 39

What is RNAi and why is it used?

Answer 39

Small interfering RNAs and microRNAs can be combined with proteins to form RNA-induced silencing complex (RISC) that cleaves mRNAs and prevents their translation

A way of silencing genes without editing the actual gene