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Nucleic Acid and Proteins, Lecture notes of Nursing

Xavier University (XU)Nursing

A lecture notes that has a explanation, bullet points, examples, summary of the topic. This consist of Blood group antigens, blood collection sets and characterization of nuclear acid.

Typology: Lecture notes

2020/2021

Available from 03/27/2023

aishy-rubin
aishy-rubin 🇵🇭

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1
MOLECULAR BIOLOGY LECTURE Finals Topic 1

CHARACTERIZATION OF NUCLEIC ACIDS & PROTEINS
We start with the Enzymes:
NUCLEASES
Enzymes that degrade DNA molecules by breaking the
phosphodiester bonds that link one nucleotide to the
next in a DNA strand
Two types of Nucleases: Exonuclease and
Endonuclease
Varying specificity
May be specific for DNA or RNA
They could also be specific for a DNA-RNA
hybrid; such as in the case of:
o RNase H cleaves the RNA strand of a
DNA-RNA hybrid
With those infos, we can infer that nucleases
have varying specificity. It is not for DNA alone
or RNA alone, but they can be also be used for
hybrids.
MECHANISM OF EACH TYPE OF NUCLEASES
1. EXONUCLEASES
Remove nucleotides one at a time from the end of the
DNA molecule.
When a nuclease hydrolyzes an ester bond in a
phosphodiester linkage, it will have specificity for
either of the two-ester   
leotides.
An exonuclease also may either attack a
 
       
Either way, it targets the end.
Examples of Exonucleases:
Intro: For when it comes to strand preference, our nucleases
may be specific for a single strand nucleotide chain, or it could
be specific for double helix, or it could be specific for both.
a) Bal31
Bal31 can remove nucleotides from both
strands of a double-stranded molecule.
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DĂ͛ĂŵďŝŐĂŝůWĂƵůĂŶ͕ZDd

CHARACTERIZATION OF NUCLEIC ACIDS & PROTEINS

We start with the Enzymes:

NUCLEASES

x Enzymes that degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand x Two types of Nucleases: Exonuclease and Endonuclease x Varying specificity 9 May be specific for DNA or RNA 9 They could also be specific for a DNA-RNA hybrid; such as in the case of: o RNase H ʹ cleaves the RNA strand of a DNA-RNA hybrid 9 With those infos, we can infer that nucleases have varying specificity. It is not for DNA alone or RNA alone, but they can be also be used for hybrids.

MECHANISM OF EACH TYPE OF NUCLEASES

1. EXONUCLEASES

x Remove nucleotides one at a time from the end of the DNA molecule. x When a nuclease hydrolyzes an ester bond in a phosphodiester linkage, it will have specificity for either of the two-ester ďŽŶĚ͘'ĞŶĞƌĂƚŝŶŐĞŝƚŚĞƌĂϱ͛ ŶƵĐůĞŽƚŝĚĞƐŽƌϯ͛ŶƵĐ leotides. x An exonuclease also may either attack a ƉŽůLJŶƵĐůĞŽƚŝĚĞĐŚĂŝŶĨƌŽŵƚŚĞϱ͛ ʹ ϯ͛ĞŶĚ Žƌ ŚLJĚƌŽůLJnjĞƐ ŝƚ ĨƌŽŵ ƚŚĞ ϯ͛ ʹ ϱ͛ ĞŶĚ͘ Either way, it targets the end.

Examples of Exonucleases:

Intro: For when it comes to strand preference, our nucleases may be specific for a single strand nucleotide chain, or it could be specific for double helix, or it could be specific for both. a) Bal 9 Bal31 can remove nucleotides from both strands of a double-stranded molecule.

DĂ͛ĂŵďŝŐĂŝůWĂƵůĂŶ͕ZDd b) Exonuclease III 9 Exonuclease III can degrade just one strand of a double-stranded molecule, leaving single- stranded DNA as a product.

2. ENDONUCLEASE

x Endonuclease will hydrolyze internal bonds within a polynucleotide chain. x In other words, it will break the phosphodiester bond at the middle or somewhere along the strand. x An endonuclease can either attack the ƉŚŽƐƉŚŽĚŝĞƐƚĞƌďŽŶĚĨƌŽŵƚŚĞϱ͛ĞŶĚŽƌĨƌŽŵƚŚĞϯ͛ end of the linkage, either of the two. x It is not at the end, it is somewhere within the polynucleotide chain.

Example of Endonucleases:

a) S1 Endonuclease 9 Cleaves only single strands of DNA b) DNase I 9 Cuts both single and double strand RESTRICTION ENDONUCLEASES x The enzyme more commonly used in the lab. x Recognize a specific nucleotide sequence and cleave the DNA molecules internally. 9 It is not random; it is specific for a particular nucleotide sequence

DĂ͛ĂŵďŝŐĂŝůWĂƵůĂŶ͕ZDd

  1. Isocaudomers x Produce the same nucleotide extensions but have different recognition sites. 9 ĂƐŝĐĂůůLJ͕ ŝƚ͛Ɛ ƚŚĞ ƉƌŽĚƵĐƚ ƚŚĂƚ ŝƐ ƚŚĞ ƐĂŵĞ͘ But the recognition site is different. x Ncol from Nocardia corallina x Pagl from Pseudomonas alcaligenes ANALYSIS OF RESTRICTION DIGESTED FRAGEMENTS x How do we apply our restriction endonucleases in the lab? Through analysis of restriction digested fragments. x Before sequencing a large stretch of DNA, some preliminary mapping is usually done to locate the cutting site and to examine the sizes of fragments (remember your DNA sequencing). The Process:
  2. Restriction Digest x First is the formation or combining what is called a restriction digest. x A restriction digest includes your: 9 template DNA 9 restriction enzyme, and 9 magnesium (Mg2+) at an optimal condition x The restriction digest results in a number of DNA fragments
  3. DNA Fragments x The sizes of which depend on the exact positions of the recognition sequences for the endonuclease in the original molecule. x The restriction digested DNA fragments (which is the product kanina) will then be analyzed through Gel Electrophoresis.
  4. Gel Electrophoresis x Gel electrophoresis allows for the separation of the DNA fragments on the basis of their site. x What happens next is we see now the migrating; and from that we will create what is called a Restriction Map.
  5. Restriction Map x A map based on the restriction sites determined by Gel Electrophoresis that represents a linear sequence of the sites at which particular restriction enzymes find their targets. x Example is the pic below. This is a restriction map of a linear sequence of the sites at which particular restriction enzymes find their targets. x These colored ones (kanang gi encircle) are the locations of the recognition sites for the restriction enzymes A & B that was used here. x What happens is like what is done in DNA sequencing, especially those of the bigger DNA sequencing, we start first with the analysis of the fragments. x So, what happens is we connect them together through a restriction map. x Next, is if we wish to identify a specific DNA region, we then analyze this digested fragment into a technique known as Southern Blot. ~ We continue on to the next enzymes on the second video.

DĂ͛ĂŵďŝŐĂŝůWĂƵůĂŶ͕ZDd To continue, we have our next enzymes. POLYMERASES x Enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template x Four types: 9 DNA polymerase I 9 Klenow fragment 9 Taq DNA polymerase 9 Reverse transcriptase Note: x DNA polymerase I 9 Prepared from E. coli 9 Has DNA polymerase activity, can attach to short single stranded region or in double stranded DNA molecule. 9 In other words, your DNA polymerase I can attach to one of the single strand region of your dsDNA. It will then synthesize a completely new strand for that particular strand. 9 /ƚ͛Ɛ ĂŶ enzyme with a dual activity; involves polymerization and degradation 9 Mild proteolytic treatment of DNA polymerase I will produce 2 fragments. 9 After proteolytic treatment, you will have 2 polypeptide, a large fragment we know as the Klenow fragment x Klenow fragment 9 >ĂƌŐĞĨƌĂŐŵĞŶƚ͖ϯ͛ - ϱ͛ĞdžŽŶƵĐůĞĂƐĞĂĐƚŝǀŝƚLJ 9 Smaller fragment (not named)- ŚĂƐ ϱ͛ - ϯ͛ exonuclease activity 9 Klenow fragment can synthesize a complementary DNA strand on a single stranded template also known as the nick region 9 Used to performed DNA end filling, when we want to connect various contigs in DNA sequencing. x Taq DNA polymerase 9 From the bacteria Thermus aquaticus 9 DNA polymerase 1 of the bacterium 9 Thermostable polymerase; suitable for the DNA polymerase reaction x Reverse transcriptase 9 An enzyme that is involved in the replication of several kinds of virus, especially your Retroviruses 9 Utilizes RNA as a template and not DNA to synthesize a complimentary DNA strand 9 Used in reverse transcriptase PCR 9 An enzyme that has an ability to synthesize a DNA strand complementary to an RNA template 9 We call these newly synthesized DNA strand as Complementary DNA (cDNA ). 9 Used to evaluate amount of RNA 9 Used in establishment of expression profile for the clinical evaluation of the change in gene expression pattern. DNA MODIFYING ENZYMES x Alkaline phosphate ( from E. coli, calf intestinal tissue, or arctic shrimp) x Polynucleotide kinase (from E. coli infected with T phage ) x Terminal deoxynucleotidyl transferases (from calf thymus tissue ) How do they modify DNA? x Alkaline phosphatase would remove the phosphate ŐƌŽƵƉƉƌĞƐĞŶƚĂƚƚŚĞϱ͛ƚĞƌŵŝŶƵƐ 9 Removed product will be replace by hydroxyl