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BIOL 200 > PCR + sequencing + cloning > Flashcards

PCR + sequencing + cloning Flashcards

1
Q

What are the necessary elements for Polymerase Chain Reaction?

A
  • DNA template
  • Taq pol (operates at high temp, comes from bacteria, no proofreading so can have errors, okk for short segments)
  • DNA pair of Primers (one complementary to 5’ end, one for 3’ end)
  • dNTPs (for amplification)
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2
Q

What are some uses for PCR?

A
  • Sequencing
  • DNA cloning (isolating particular gene)
  • Detection of pathogens
  • Gene editing
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3
Q

What does PCR rely on?

A

It relies on knowing the nucleotide sequence at the ends of the region to be amplified

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4
Q

What are the steps of PCR?

A
  1. Heat solution to 95˚C : Denature double helix (2 strands come appart, breaks H-bonds)
  2. Cool to 50˚C: primers anneal to DNA strands (excess amount of primers in initial solution)
    2 types of primers: 3’ to 5’ and 5’ to 3’
  3. Raise T˚ to 70˚C: DNA synthesized with taq pol
    (1st cycle synthesizes more than the sequence, but after segment ends so all good)

Repeat 1-3 for 40 cycles

  1. Final DNA extension: cool to bring single strands into double stranded helix again
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5
Q

How are produced oligonucleotides?

A

oligonucleotides = primers used in PCR

Designed by computer to be complementary and specific to end of sequence to be amplified + commercially synthesized

Computers make sure no other similar sequence in gene, have abt same ratio of AT/CG for both primers as want same T˚ of denaturation

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6
Q

What is the Dideoxycytidine’s Chain-Termination Method of DNA sequencing method (Classical Sanger Sequencing)?
Not so used anymore

A

4 tubes with:
DNA Polymerase
Oligonucleotide primer
DNA template
dNTPs (100 mM) of all 4 nucleotides

Each tube has 1 chain terminator:
ddATP (1mM)
ddGTP (1mM)
ddTTP (1mM)
ddCTP (1mM)
*has H on 3’ instead of OH so can’t make phosphodiester bonds

Use gel electrophoresis to figure out sequence

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7
Q

What are the limitations of Sanger sequencing?

A

Polymerase only runs for 300-500 nucleotides
Gells can only resolve this much so can only sequence short sequences

Rate of sequence of production limited by total number of reactions that can be performed at one time

Can only run about 40 cycles before lose too much precision

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7
Q

How does Next-Generation Sequencing (NGS) work?

A

*Single sequencing instrument carries out millions of sequencing reactions

One type:
1. Ligating linkers to mixture of DNA
2. Denature, then anneal to primers on solid support
PCR conducted to amplify DNA fragments in fixed spacial arrangement

  1. Double stranded DNA cut and only 1 strand sequenced with fluorescently labeled dNTPs (different colour/base)
    For multiple DNA sequence at the time:

a. Add primer
b. Add fluorescently labeled dNTP which binds to single strand (just beside the primer)
c. Fluorescent imaging to determine which dNTP bound to which fragment
d. Chemically remove fluorophore (keep base pair)
e. Repeat until DNA strand is replicated, each base adds to next place beside

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8
Q

How is the whole genome assembled when doing Next-Generation Sequencing?

A

Computational models find overlaps in the fragments and align them to form a complete genomic sequence

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9
Q

What step is skipped in even newer sequencing methods than NGS?

A

PCR (sequencing is done on a single molecule)

motor protein moves through the DNA and send an electric signal to each base pair
Called nanopore

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10
Q

What are the advantages of Nanopore?
DNA sequencing technique with electric current (single molecule needed)

A
  • Sequencing single molecules opens possibility of studying new biological questions
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11
Q

What are the general steps of the recombinant DNA technology?

A
  1. Vector + DNA fragment
  2. Recombinant DNA
  3. Replication of recombinant DNA within host cell
  4. Isolation, sequencing and manipulation of purified DNA fragment
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12
Q

What are plasmids?

A
  • They are the most common vector used in recombinant DNA technologies
  • Circular
  • double-stranded DNA
  • extrachromosomal (replicate independently from cell replication and division)
  • found in bacteria and lower eukaryotes
  • replication of plasmids occurs before cell division (not same amount of plasmids in every cells)
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13
Q

What are the roles of restriction enzymes (restriction endonucleases)?

A

Since the vector is ciruclar, have to cut it to insert DNA fragment, they cut phosphodiester bonds in vector (symmetrical fashion)
*Staggered cut to have sticky ends (not all rest. enzymes do, but better for further ligation)
Recognize a specific DNA sequence (Usually the sequence is a palyndrome to have same sticky ends)

Want 1 recognition site/vector

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14
Q

What is a polylinker?

A

It is a sequence with recognition sites for many different restriction enzymes
If sense of the DNA fragment matters, use 2 different restriction enzymes to have different sticky ends on each side
Polylinker = site where the exogenous DNA can be inserted

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15
Q

What are the 3 important parts of a Plasmid vector?

A
  • Polylinker/recognition site
  • Origine site
  • drug resistant segment
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16
Q

What is the process of transformation of a Recombinant plasmid?

A

Process by which we isolate the transformed cells with recombinant and kill other cells
1. Mix E. coli with plasmids in presence of CaCl2, heat pulse (Recombinant plasmids entre the cell)
2. Culture on nutrient agar plates with ampicillin (cells without recombinant vector die)
3. Plasmid replication (before and independently from chromosome replication)
4. Cell multiplication (each cell has a different # recomb. vectors

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17
Q

What are DNA libraries?

A

Where permamnent collections of genes can be obtaied and maintained, collection of Recombinant vectors with different segments in them
- genomic libraries (chromosomal DNA, all DNA in the genome, mutliple different little segments?)
- cDNA libraries (represent mRNA present in a given sample)

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18
Q

How are cDNA libraries produced?

A

from RNA using reverse transcriptase (protein → RNA ← cDNA)

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19
Q

By which process are DNA libraries produced?

A
  1. BamH1 cuts vector (plasmid of phage)
  2. Sau3A partially digests genomic DNA
  3. both are ligated together to form a recombinant DNA
    *4-bases sticky ends connect with central 4-bases of BamH1 (longer segment) sticky ends
20
Q

How does generation of cloning of a cDNA copy of mRNA works? (reverse transcription)

A

cDNA doesn’t contain whole genome only specific functional parts for specific reactions wanted
1. oligo-dT primer hybridize with mRNA (with 3’poly(A) tail)
2. reverse-transcribe RNA into cDNA
3. single DNA strand reverse transcribed
4. 2nd strand synthesized
5. EcoR1 linkes ligated to cDNA on both ends of segment (on AT end and GC ends)
6. Cleave with EcoR1 (staggered cut makes sticky ends)
7. Ligate to plasmdi (with also EcoR1 sticky ends)
8. Transform E. coli (put recomb in E coli + select for ampr)

21
Q

What uses can be made of a recombinant DNA construct? Explain

A
  • Microarray (reveal mRNA expression/their level, co-regulation)
  • In situ hybridization (localization of where certain genes are expressed)
  • enable reugulated expression of exogenous genes + production of proteins in prokayotic and eukaryotic cells
22
Q

How does microarray analysis work?

A

Technic to study expression of thousands of genes simultaneously (expression under specific conditions)
A microarray = microscope slide + thousands of spots containing different sepcific known probes (genes) (to detect DNA expression)
add specific condition (ex: serum) and see which color is present (if green, gene expression decreases with serum bc green was solution without serum, if red, opposit)
in incorporated solution, there are many DNA segments which recognize the genes on the microarray plate

23
Q

What is the lac promoter? What is used for?

A

Used to produce eukaryotic proteins in E. coli and treat E. coli with lactose
Lac promoter = inducible (can be turned on and off in different environments)
When add IPTG, lac promoter is induced —> G-CSF cDNA translation to mRNA is promoted—> G-CSF protein is synthesized

In the vector, the lacZ gene (which comes with the lac promoter in the original vector) is replaced by G-CSF (regulator) cDNA, then the E coli is tranformed (recomb. DNA added to it + drug selection)
Then, G-CSF mRNA in trascripted from cDNA and G-CSF protein is produced in the cell

24
Q

What is the genome?

A

The entirety of an organism’s hereditary information
Usually DNA, but some viruses have RNA genomes

What is the measurement unit of dsDNA?
base-pairs
1000 bp = 1 kilobase
1 000 000 = 1Mbp
Biological complexity is NOT related the number of base-pairs

24
Q

What are 2 ways genes can be expressed in cultured animal cells (instead of in bacteria)?

A

In both cases, the recomb. vector has promoter, cDNA
1. Transient transfection (with viral origin of replication):
Transfect cultured cells by lipid treatement or electroporation
Protein is expressed from cDNA in plasmid DNA
Plasmid doesn’t go into chromosomes, no selection so eventually gets lost

  1. Stable transfection (with drug resistant segment):
    Transfect cultured cells by lipid treatement or electroporation
    Select for G-418 resistance
    Protein is expressed from cDNA integrated into host chormosome (replicates with cell)
25
Q

How many specied have been sequenced? What is the largest sequenced genome?

A

3278 species = 0.2% of animals
Lagest sequenced genome = 43 Gb (14x human genome), the Australian lungfish Xiphophorus has a lot of transposable elements (not more genes than other vertebrates)

26
Q

How large is the human genome?

A

3300 Mb

27
Q

What is cluster analysis? What is useful for?

A

Used to identify coordinately regulated genes (in specific envrionments)

28
Q

What are retroviral vectors?

A

They can be used to integrate cloned genes into mammalian genome (into tissues/organs of the living organism)
Tissue culture cells transiantly infected with 3 different plasmid cells
- Vector plasmid —> LTR + cloned gene + neor + LTR (long terminal repeat)
- Packaging plasmid (includes a lot of viral proteins,
- Viral coat plasmid (encodes a protein that’ll target the virus to particular cell type that you want to tranfect with gene of interest) —> has a promoter + Gene for VSV G protein + viral origin of replication

So the cell that get all 3, can make functional virus particles that have cloned gene of interest into the viral genome
Can be targetted to a particular tissue type
Lentivirus = type of virus usually made

Can then infect living targetted tissues —> can introduce correct gene into tissues that could have a mutation

29
Q

What is the definition of a gene?

A

The entire nucleic acid sequence that is necessary for the synthesis of a functional product (polypeptide or RNA)
A lot of genes encode RNAs that have other functions than coding for proteins (ex: ribosomal RNA)
Genes can be considered as a transcription unit

30
Q

What is contained in a simple transcription unit?

A
  • Control regions = promoter and cis-regulatory factors, 50 kb between a and b (2 control regions)
  • Exons (contains coding info necessary to produce functional protein) —> coding regions/Open Reading Frame
  • Introns (in eukaryotes?) = non-coding regions that separate exons, are spliced out during mRNA processing
  • Poly(A) site
    *mature mRNA only has exons
31
Q

What can be found using BLAST?

A

Nucleic acid and protein sequence similarities between proteins with similar function
bc encode for functional domains

32
Q

How does the number of proteins, the gene density vary between species?

A

Protein number doesn’t vary a lot (much less than DNA content/size)
Gene density = much greater in lower eukaryotes than in more complex eukaryotes

33
Q

What is a pseudogene?

A

It has the structure of a gene in DNA but has stop codons and other features that make it non-functional

34
Q

What are orthologs and paralogs?

A

Orthologs = same protein in different species (ex: alpha-tubulin in humans and in flies)
Paralogs = closely related proteins in the same species (a-tubulin and b-tubulin in humans)
*Can illuminate evolutionar relationships

35
Q

What is a gene family? What is a solitary gene?

A

25-50% protein-coding genes = solitary/single-copy genes = represented once in the genome

Rest occur as duplicate or multiple copies, set of related genes formed by duplication of an orginigal single-copy gene = gene family

36
Q

What is the most common type of simple-sequence repeats?

A

Microsatellite DNA

37
Q

What are Microsatellite DNA?

A
  • The most common type of SSR
  • 1-4 bp in length
  • Arrays up to 600 bp composed of tandem repeat units
  • Sometimes found in transcription units (not always in non-coding RNA)
  • expansion underlie several neuromuscular diseases —> myotonic dystriphy and spinocerebellar ataxia
38
Q

By what can short repeated sequences be generated?

A

Short repeated sequences can be generated by backward slippage during replication (DNA replicates the sequence n+1 times be it forgets it did on of them and goes back on the template to the start of the sequence it just translated)

When a repeat sequence exceeds the normal range and is linked to a disease, start seeing the effects of the disease.

39
Q

What are minisatellite DNA?

A

A type of simple-sequence repeat

  • Repeat units = 14 -100 bp in length
  • 20 - 20 tandem repeat units
  • Arrays of 1-5 kbp in length
  • Often in centromens and telomeres (non-coding regions)
40
Q

What are repeated sequences used for? and Why?

A

They are used for paternity determination and to identify criminals bc they vary and length so distinguish different repeat sequences in different individual by gel electrophoresis

Genetically passed on (in the chromosomes)

41
Q

What are tranposable (mobile) DNA elements?

A

Transposable DNA elements are SSR that move within genome by different mechanisms

  • influenced evolution
  • can cause mutations leading to disease
42
Q

What are the 2 major classes of transposons?

A

DNA transposon: (3% of human genome)
- mvt entirely DNA based
- transposon leaves its former site to go in other site (no copy made)

Retrotransposon: (40% of human genome)
- mvt involves RNA intermediate
- Retrotransposon stays in former site (doesn’t leave)
1. RNA pol synthesizes an RNA intermediate
2. Reverse transcriptase makes DNA intermediate that will be inserted in the target DNA

43
Q

Since DNA transposons don’t create copies when they move, how can they increase in copies/number in DNA?

A

A DNA transposon moves from a region that has been replicated to one that has not on the parent DNA
Will add a copy in the daughter chromosome (DNA transposon just changed place in the parent chromosome)

44
Q

What is the general structure of eukaryotic LTR retrotransposons?
Explain the segments

A

LTR = long terminal repeats (250-600 bp)

Protein coding regions = encode reverse transcriptase (for RNA intermediate), integrase, and other proteins (that allow the DNA segment to be inserted into a future target DNA)

Structure of LTR retrotransposon = Target-site direct repeat (5-10 bp) + LTR + protein-coding region + LTR + target-site direct repeat

45
Q

How does generation of retroviral genomic RNA from integrated retroviral DNA occur?
In other word, how does the DNA in coding region of integrated retro-virus becomes retroviral RNA genome ?

A
  1. Integrated retroviral DNA → Primary transcript (by RNA pol II)
  2. Primary transcript → Retroviral RNA genome (by RNA-processing enzymes + poly(A) pol)
    Retroviral genome = R-U5-coding region-U3-R-(A)n
    *LTR = U3-R-U5
    Poly(A) synthesizes from poly(A) site in the LTR betwee U3-R and U5
46
Q

What is a retro-virus?

A

A viruse whose genome ressembles an LTR retrotransposon

47
Q

What are LINEs?

A

LINE = long interpreted element (abt 6kb)
Nonvrial DNA retrotransposons (can not be packaged to a virus)

Structure: Target-site direct repeat + AT-rich region + protein coding region + AT-rich region + Target-site
*No pol(A) site

Protein coding region = ORF1 + ORF2
Open Reading Frame 1encodes RNA binding protein involved in nuclear transport of LINE RNA

Open Reading Frame 2 encodes for reverse transcritpase + a nuclease

There are 900,000 in human genome (54Mb, approx. 2% of total DNA)

BIOL 200 (11 decks)

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